This work was funded by the Department of Defense (W81XWH-13-01-0243) and the National Institutes of Health (R37 AG008796). We thank Alan Baker in Northwestern University&#39;s machine shop for building the head-fixed cylinder apparatus. We thank Dr. Shoai Hattori for his guidance in MATLAB and Solidworks. We thank Dr. John Power for the LabView software that controlled the experiment.

All procedures involving mice were performed in accordance with protocols approved by Northwestern University&#39;s Institutional Animal Care and Use Committee based on guidelines issued by the National Institute of Health.
1. The Cylinder (Figure 2A)
<ol>
	<li>Construct the cylinder as described by Chettih et al. and Heiney et al. from a long foam cylinder14-15. 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 (e.g., Plexiglas)15.</li>
	<li>Erect two vertical metal rods of 12.7 mm in diameter on either side of the cylinder.</li>
	<li>Attach two right-angle mounts onto the metal rods.</li>
	<li>Secure two more rods (5 cm long, 12.7 mm in diameter) through the right-angle mounts. Bevel the ends of these rods and level their ends above the center of the cylinder (Figure 2G).</li>
	<li>Drill and tap a hole through the leveled ends of the rods to fit two 4-40 machine screws that will hold the wings of the connector in place (Figure 2G).</li>
</ol>
2. Assembling the Whisker Stimulation System (Figure 2B)
<ol>
	<li>Cut a 10-tooth segment of comb from a regular hair comb.</li>
	<li>Cut a slit about 2-3 mm wide and about 5 mm deep on the top part of the comb to accept a 2-layer rectangular bending actuator.</li>
	<li>Solder one wire to each surface of the bending actuator. Use the solder and flux provided by the manufacturer. Cover the actuator and solder with electrical tape to protect the mouse and user from the electrical current.</li>
	<li>Slip the actuator strip into the slit cut into the comb so that the bending actuator strip sits perpendicular to the plane of the comb. Angle the comb so that it is tilted at 45&#176; and more naturally follows the curve of the mouse&#39;s face.</li>
	<li>Secure the end of the actuator strip to the top of the comb with quick-setting epoxy. 
	Note: The wires soldered to the bending actuator strip should be connected to a rheostat that delivers 40 volts to the actuator. Previous experiments have shown that 40 volts is effective for conditioning but does not evoke any startle responses.</li>
	<li>Place the bending actuator and the comb on a moveable mount. A pipette holder attached to a magnetic base as normally used for in vitro electrophysiology rigs works well.</li>
</ol>
3. Assembling the Connector that Mounts to Rods above the Cylinder (Figure 2C, 2E)
Note: The connector is a 3-D printed 7-hole strip modeled from the Amphenol 221 series nylon strip used for the tether by Weiss and Disterhoft and Galvez et al.10,17
<ol>
	<li>Tap the first hole in the strip for a 0-80 x-1&#34; machine screw. This will serve as a locking screw to anchor the headpiece to the connector. Affix a 0-80 nut to the head of the locking screw with cyanoacrylic glue in order to facilitate turning the screw by hand.</li>
	<li>Leave one hole empty after the tapped hole (to allow room for the 0-80 machine nut) and push five gold-plated relia-tac sockets through the remaining five holes.</li>
	<li>Strip the coating off five long wires (wires obtained from an Ethernet cable) and solder to the ends of the sockets. 
	Note: The first two wires will be used to record the electromyographic (EMG) signals that will detect an eyeblink response following signal filtering and amplification from an amplifier. The second two will deliver the shock signal from a stimulus isolator. The last wire will serve as a ground connection. These wires will be connected to the systems used for the timed CS-US delivery.</li>
	<li>Ensure electrical connectivity between the wires and the sockets with a multimeter. Put the multimeter on the continuity setting and hold one of the probes to the pin and the other probe to the stripped ends of the wire. The pins and the wires are electrically connected when the resistance is low and the multimeter emits a tone.
	<ol>
		<li>Alternatively, if the multimeter does not have a continuity setting, measure the resistance between the pins and the wires. There is electrical continuity if there is very little resistance measured.</li>
	</ol>
	</li>
	<li>Prepare the wings of the connector from two TO-220 style transistors. Cut off the emitter, base, and collector leads of the transistor and level the surface so it can be affixed to the connector.</li>
	<li>Cut the edge of the mounting tab to breach the hole so the tab can slip underneath the head of the screw.</li>
	<li>Epoxy the transistors to the connector. The hole in the mounting tab will be used to secure the connector to the rods above the cylinder.</li>
	<li>Apply epoxy to the base of the wires protruding from the sockets and allow the epoxy to dry. The epoxy will insulate and protect the connections.</li>
</ol>
4. Preparing the Headpiece (Figure 2D, 2E)
Note: The headpiece strip is a 3-D printed 7-hole strip modeled from the Amphenol 221 series nylon strip used for the headpiece by Weiss and Disterhoft and Galvez et al.10,17. This piece is no longer manufactured commercially. The printer file can be downloaded from this journal&#39;s website.
<ol>
	<li>Tap the first hole in the strip for a 0-80 x 1&#34; machine screw and leave one hole empty after the first hole.</li>
	<li>Push five gold-plated pins through the bottom of the remaining five holes (through the narrower ends). Use a vise to facilitate pushing the pins into the strip evenly.</li>
	<li>Using a thermal stripper, remove about 0.5 cm of polyimide coating off the end of a stainless steel wire (0.005 in. diameter), solder the stripped end of the wire to the opening of one of the pins and cut the wire to 0.6-0.7 cm.</li>
	<li>Strip about 0.2 cm from the end of the wire to allow current to pass to the animal.</li>
	<li>Repeat steps 4.3-4.4 for the remaining three wires. 
	Note:The first two wires will record EMG responses while the second two will serve to deliver a shock to the animal.</li>
	<li>As the last (fifth) wire will serve as a ground wire, cut about 5 cm of uncoated stainless steel wire (0.005 in. diameter), and solder to the remaining pin.</li>
	<li>Ensure continuity between the wires and the pins with a multimeter. (In the case of the ground wire, which is uncoated, the multimeter probe can be placed anywhere along the wire.)</li>
</ol>
5. Surgical Preparation (Figure 2F)
<ol>
	<li>Sterilize all surgical tools, including the headpiece. Sterilize the headpiece by soaking it in alcohol and then rinsing with sterile saline.</li>
	<li>Anesthetize the mouse in an induction chamber with 3-4% vaporized isoflurane mixed with a flow rate of 1-2 L of oxygen per minute.</li>
	<li>Ascertain that the animal is fully anesthetized with a toe pinch. The mouse is fully anesthetized when it does not respond reflexively to the toe pinch.</li>
	<li>Once the animal has been fully anesthetized, inject a dose of buprenorphine hydrochloride as an analgesic (0.05&#8211;2 mg/kg, sc), shave the top of the animal&#8217;s head, place it on a covered heating pad on the base of the stereotaxic frame, ascertain that the animal is fully anesthetized, and secure the head to the frame.&#160;Maintain flow of vaporized isoflurane to the animal, switching to 2% at a flow rate of 1-2 L of oxygen per minute. Apply a small amount of ophthalmic ointment to the corneas.</li>
	<li>Disinfect the scalp with povidone-iodine and alcohol three time each, alternating between the two.</li>
	<li>With a number 10 or 15 scalpel blade, make an incision along the midline of the scalp, exposing the skull from the front of the eyes to past the interparietal bone (~1.5-2 cm).</li>
	<li>Hold back the flaps of the skin with micro clips. Place one clip above the eye, one along the middle of the rostral-caudal axis, and one at the back of the skull bilaterally (i.e., use six clips in all). In order to prevent headpiece avulsion, expose as much of the skull as possible, including the sides and back. This will increase the surface area for the application of the adhesive luting cement.</li>
	<li>Using the scalpel, scrape along the top of the skull to remove the periosteum and ensure a clean and dry working surface. Clean the top of the skull with 3% hydrogen peroxide three times.</li>
	<li>Drill two holes into the skull with a size 34 inverted cone burr or a 1.6 mm engraving cutter drill bit to accept 00-90 screws (0.0625 in. long). The screws will provide the electrical ground connection. Place one hole in front of Bregma, and the other in front of Lambda, left of the midline when conditioning the right eye. Place one screw in each hole. Lower the screw 0.28 mm into the skull for each full turn; two full turns is sufficient.</li>
	<li>Create several small divots of approximately 0.75 mm in diameter on the skull in order to increase the surface area and grip strength for the cement.</li>
	<li>Take the completed headpiece and wind the ground wire in a figure-eight configuration around the two screws. Allow some slack (~1.5 cm between the headpiece and the screw) in the ground wire so the headpiece can be positioned properly later. 
	Note: The figure eight usually ensures a good electrical connection between the wire and the ground screws. The wire can also be soldered to the screw to ensure an electrical connection.</li>
	<li>Apply the adhesive luting cement. Follow the manufacturer&#39;s instructions for mixing the adhesive luting cement or mix 4 scoops of L-Powder mix, 8 drops of the base, and 2 drops of the catalyst in a cold ceramic mixing dish (ensure the temperature strip on the mixing dish is completely black.) The cold temperature extends the working time of the cement.
	<ol>
		<li>Coat the skull and the screws liberally with the luting cement and allow the cement to dry. This should take only a few minutes.</li>
	</ol>
	</li>
	<li>After the cement has dried, position the headpiece vertically, with the pins standing up, above the skull. Hold the headpiece in place with a holder similar to the connector 
	Note: See &#34;3. Assembling the Connector&#34;-The holder need only be a five-hole strip with gold-plated sockets in order to receive the pins of the headpiece. The holder is attached to an arm on the stereotaxic frame. The use of the holder and arm facilitates the positioning of the headpiece and the wires.</li>
	<li>After the headpiece has been positioned, peel back the skin around the periorbital area and position the two shock wires to allow the 0.2 cm stripped end to rest underneath the skin and approximately 2-4 mm directly caudal to the eye. Do not allow the ends of the two wires to touch each other. Position the two EMG wires on the muscularis orbicularis oculi above the eye socket.
	<ol>
		<li>If necessary, cut off the ends of the wires if they are too long and seem as though they may directly scratch the orbit and result in infection. If cutting off the ends of the wire, ensure that there is enough bare wire exposed. Alternatively, if the wires seem too long, bend the wire back at the base, where they extend out of the headpiece.</li>
		<li>Cement the base of the wires (i.e., the end near the headpiece) in place on the skull with a small spread of the adhesive luting cement and allow to dry (use half of the portion described earlier).</li>
	</ol>
	</li>
	<li>Remove the micro clips and gently fold the flaps of skin back over the cement. Allow the skin to settle naturally to prevent tension on any part of the skin in order to avoid distortion of the eyelid, prevention of the eyeblink response, and distress to the animal.</li>
	<li>Seal the exposed area with dental cement, covering everything from the incised skin to the edge of the top of the headpiece with cement. Take extra care to avoid dripping cement on the eyes or the pins of the headpiece. Partially cured cement can be smoothed and manipulated with a cotton swab that is dampened with dental cement solvent. Allow the cement to dry completely before removing the headpiece holder.</li>
	<li>Allow the animal to recover from anesthesia on a warmed surface before replacing the animal back in its cage.&#160;Administer Metacam (1 mg/kg, SQ) and then place the animal back in its cage.</li>
	<li>Allow the animal 5-7 days of recovery before testing or training. Maintain standard post-operative care to the animal according to your institution&#39;s guidelines.&#160;The animal should be checked at least once per day after it has recovered from surgery. Watch for signs of poor eating and/or drinking and listless behavior. If pain is suspected, provide Metacam every 24 hr&#160;(same dose as at the end of post-surgery) until alleviated. Lidocaine can be locally applied to the wound if the animal is scratching or shows signs of discomfort.</li>
</ol>
6. Placing the Mouse on the Cylinder and Training (Figure 2G)
<ol>
	<li>To place the mouse onto the cylinder, restrain the mouse with a hand briefly by holding it by the tail with one hand and then grasping the mouse behind the shoulders with forefinger and thumb with the other. Wrap the remaining fingers around the mouse&#39;s abdomen and torso.</li>
	<li>With the hand not restraining the mouse, attach the connector to the headpiece on the mouse&#39;s head and turn the locking screw.</li>
	<li>Place the mouse gently on the cylinder and hold it in place while attaching the connector to the frame. Use the two screws to fasten the connector to the bars above the cylinder.</li>
	<li>Once the connector has been secured to the cylinder frame, release the hold on the mouse. Give the mouse one day of two sessions of habituation to the cylinder. Allow each habituation session the same duration as each conditioning session.
	<ol>
		<li>Record spontaneous blink rate during habituation and pre-expose the mice to the whisker vibration CS in order to minimize the occurrence of startle responses. Apply the CS as during an actual conditioning session, but without the electrical shock US.</li>
		<li>Apply the whisker stimulation CS by placing the Piezo system close (about 0.5 cm) to the conditioned side (the right side) of the mouse, while slipping the teeth of the comb over individual whiskers. Ensure that the same whiskers are stimulated day to day by placing the Piezo system in the same position from day to day.</li>
	</ol>
	</li>
	<li>Begin training the mouse on classical conditioning. Deliver two sessions of training per day for five days. Leave about two hours between each training session.
	<ol>
		<li>To the conditioned group, deliver thirty trials per session of a 250 msec long whisker vibration CS (60 Hz) paired with a 100 msec long electrical pulse US (0.3 mA - 3 mA, delivered from a WPI A385R stimulus isolator). Separate the CS and the US with a 250 msec long stimulus-free trace interval to make the task dependent on the hippocampus (Figure 1)23.</li>
		<li>Adjust the intensity of the electrical shock for each animal so that a blink is evoked. Separate trials with a random intertrial interval of 30-60 sec (average of 45 sec, total duration of 30 min per session.)</li>
		<li>Deliver thirty unpaired CS alone and US alone trials each (for a total of sixty trials) to the pseudoconditioned group, using the same stimulus parameters as stated for the conditioned group. Ensure that the CS alone and US alone trials are pseudorandomized such that no stimulus is presented more than twice in a row. Separate each trial with an average intertrial interval of 22.5 sec.</li>
	</ol>
	</li>
	<li>Deliver background noise at 65 dB throughout habituation and training in order to mask any noise of the vibration generated by the whisker stimulator.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infragranular+barrel+cortex+activity+is+enhanced+with+learning.">Infragranular barrel cortex activity is enhanced with learning.</a> J Neurophysiol. 108 (5), 1278-1287 (2012).</li></ol>

The authors declare that they have no competing financial interests.

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 Siegel et al. offers several advantages, such as allowing for certain procedures and experiments to be performed during eyeblink conditioning that had not been possible or had been limited in the past (e.g., in vivo two-photon calcium imaging)14-16. This type of preparation also facilitates the use of other techniques such as in vivo electrophysiology with tetrode arrays. As mice appear to become distressed from restraint, this head-fixed preparation was developed to keep heads secure while reducing the amount of restraint required. While this preparation allows mice to ambulate on a cylindrical treadmill, others have also successfully collected behavioral data from head-fixed mice on a stable platform24.
There are, however, certain issues that arise with the use of the head-fixed cylinder system. One concern is that when one initially places an untrained mouse on the cylinder, it may need some time to learn to rest, walk, or run on the novel apparatus. Most mice learn quickly to rest, walk, or run on the cylinder comfortably. Two habituation sessions help to accustom the mice to the cylinder and by the middle of the second session, most mice are comfortable on the cylinder. Another concern that arises is headpiece avulsion. There has been some problem with this in the past during multi day training sessions, but the use of the adhesive luting cement has ameliorated issues with headpiece avulsion. The use of the adhesive luting cement has resulted in 0% headpiece avulsions from more than 50 surgeries since using this technique. With correct use and sufficient spread of the cement on the cranium, researchers should encounter no issue with headpiece avulsion. Finally, by presenting the CS during habituation sessions, there may be some concern about latent inhibition. It is clear, however, that the mice were conditioned successfully (see Figure 7). Note that the intensity of the whisker vibration can be modified by changing the amount of voltage being passed by the rheostat. If the mice are unable to learn, the voltage can be increased to support successful conditioning while also minimizing startle responses. Researchers can also change the type of electrical shock US that is delivered to the animal. The electrical shock US can be a single electrical pulse lasting 100 msec as reported here. Alternatively, as the stimulus isolator utilized in this protocol is capable of transmitting biphasic pulses, researchers can also employ 120 Hz 6 biphasic pulse pairs at 1 msec per pulse for an effective electrical shock US as well.
Boele et al. indicated the likelihood that certain &#34;conditioned responses&#34; may arise from startle (alpha) responses to the CS or short latency responses (SLR)25. Alpha responses are considered to be rapid eyelid closures within a 50 msec latency after CS onset. Mice often keep their eyes closed for the duration of the trial, following this alpha response. SLRs, on the other hand, have a latency of 50-70 msec from CS onset and typically arise after only a few CS-US pairings. The short latency to onset of both alpha and SLRs and the fact that SLRs can arise after only a few CS-US pairings indicate that they are likely not driven by the hippocampal-cerebellar circuitry. These observations reviewed by Boele et al. therefore beg the question of whether the CRs recorded here are learned responses. As Figure 7C and 7D show, neither alpha responses nor SLRs account for a majority of the CRs recorded, suggesting that the adaptive CRs are mediated by the forebrain and cerebellum. The histograms in Figure 6 which show latency to the peak of the eyeblink response following CS onset also indicates that while certain trials may include an early onset alpha or short latency response, with more training trials, the peak of the eyeblink response actually occurs later and closer to the onset of the US, indicating the development of well-timed conditioned responses. As mentioned previously, whisker stimulation and shock intensity were attenuated to a level that was not startling or overly aversive, but remained effective in producing adaptive conditioned responses. Finally, as Figure 7B shows, the size of the CR (measured as the area under the curve in the rectified and integrated response, as in Figure 5) is larger in the conditioned group than in the pseudoconditioned group once the conditioned group reached a learning criterion of 60%, signifying that these responses require several sessions to learn, unlike SLRs and alpha responses.
Boele et al. also pointed out that EMG measurements, while an accurate method of blink detection in larger animals such as rabbits, are not as practical in small rodents such as mice25. They recommend the use of the magnetic distance measurement technique (MDMT) to avoid the detection of false positive signals that EMG recording may indicate. The MDMT technique, while impressive in its sensitivity and quality of blink detection, also presents the disadvantage of having to anesthetize the animal each day of training in order to attach the chip26. This in itself may confound learning rates. We find that the EMG signals recorded with the procedures described here are easily obtained, of high temporal resolution, reliable and relatively easy to measure and analyze.
Figure 4 shows a representative EMG signal detecting blinks with a corresponding signal from an infrared reflective optical sensor placed close to the mouse&#39;s eye during training. There is a clear correlation in blink detection between the EMG signal and the optical sensor, denoting the accuracy of blink detection using EMG recordings. An advantage of EMG recordings is that it allows for the greatest temporal resolution. Although the resolution is degraded by integrating over a 10 msec time constant, one can also analyze the raw EMG data to detect eye blink activity. Spike count is one parameter that may be used to detect CRs18. One disadvantage with EMG recordings is that signals will undoubtedly be contaminated by the electrical artifact coming from the electrical shock US (see Figures 3 - 5). This, however, does not impede the ability to record conditioned blink responses, i.e., those that occur prior to onset of the US. Another disadvantage in employing EMG recordings is that, using the present criterion for CR detection, a noisy baseline may hide what may otherwise have been detected as a CR.
The head-fixed preparation described here is similar to that presented by Heiney et al.15 There are, however, certain notable differences from their elegant system. For example, the method described here for recording eyeblink responses are wires placed subdermally above the eye to record EMG activity. This technique allows for reliable and stable recordings of eyelid activity and thus, of the eyeblink response. Proper placement and fixation of these wires ensures quality recordings that last for at least two weeks, the duration of the experiment. An advantage of using EMG wires rather than a high-speed camera as used by Heiney et al. is that EMG recordings have extremely high temporal resolution, and do not require the daily positioning and calibration that a camera requires15. The camera does, however, offer direct visual determination of eyelid closure.
Another difference between the two systems is the method for CS delivery. This particular head-fixed mouse preparation utilizes whisker stimulation as the conditioned stimulus. The results demonstrate that mice can be conditioned effectively with whisker stimulation as the CS, just as head-fixed rabbits can be conditioned with whisker vibration27. Heiney et al. demonstrated stimulation of the whisker pad as an effective CS by directing a weak puff of air to the whisker pad15. Although both techniques demonstrate effective conditioning, placing a comb over selected whiskers and vibrating the comb allows for the ability to stimulate individual whisker rows or even individual whiskers. This technique has been used in previous studies to allow for animals to serve as their own control (i.e., the stimulated whisker row was compared to a row of unstimulated whiskers)20.
In summary, a head-fixed preparation for whisker-signaled eyeblink conditioning allows for a secured cranial platform for performing advanced techniques and experimentation that had previously been impossible or difficult to perform. Mild whisker stimulation was used as the conditioned stimulus and a mild electrical shock was used as the unconditioned stimulus. Eyeblink responses were recorded with wires placed subdermally behind the eyelid. Reliable learning was demonstrated in mice with direct stimulation of selected whiskers as the CS and periorbital electrical shock as the US, and no learning was evident in mice that were given random presentations of the CS and the US. The EMG recordings provided a dependable and relatively simple method for recording eyeblink responses and observing how the responses changed across training sessions.

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 to associate the CS with the US. This learning manifests itself in the form of a conditioned response (CR), an eyeblink elicited by the CS alone that precedes the presentation of the US.
Eyeblink conditioning in the trace form includes a stimulus-free interval of a few hundred milliseconds that separates the CS and the US (Figure 1). Trace conditioning is a form of declarative learning since it requires awareness of the stimulus contingencies1. The temporal gap requires the animal to keep a neural &#39;trace&#39; of the CS in forebrain regions such as the hippocampus in order for the US and the CS to become associated1-6. Along with the forebrain regions, trace conditioning is also dependent on the cerebellum7.
Eyeblink conditioning is, therefore, a useful paradigm for the investigation of the multiple facets of memory, including acquisition, consolidation, and retrieval. During eyeblink conditioning, a control group of animals is presented with unpaired stimuli in random order to test for pseudoconditioning or sensitized responses to the CS that may be caused by US presentation alone rather than a learned CS-US association.
A commonly used apparatus for the investigation of eyeblink conditioning in rodents is a chamber in which the rodents are allowed to move about freely during the training process8-10. With this type of apparatus, a tether is normally attached to a headpiece that is affixed to the rodent&#39;s skull. The tether allows for the delivery of the US (and sometimes the CS) and for transmitting the animal&#39;s response to those stimuli (i.e., the eyeblink response)10. The tether itself may be modified based on the type of stimuli delivered and how the eyeblink response is recorded.
The reason for using &#34;freely-moving&#34; tethered mice for eyeblink conditioning is that mice struggle against restraint. Though other species may be more amenable to restraint, the major advantage of using mice in eyeblink conditioning experiments is that the majority of available genetically modified mutant strains are mouse strains. In addition to struggling, complete restraint of mice results in acute distress. A head-fixed mouse preparation that minimizes stress would increase the physiological information that can be obtained during eyeblink conditioning. For example, this system would allow imaging of cortical neurons with 2-photon microscopy11.
Head-fixed preparations have been used in previous experiments for optical imaging of the cortex through removable cranial implants, in vivo electrophysiological recordings of the rodent brain with tetrode arrays, two-photon calcium imaging, and also as a platform for eyeblink conditioning in mice11-16.
In the head-fixed system, reliable stimulation and recordings are ensured without complete restraint of the mouse (Figure 2). A headpiece like the one used in the freely moving system is affixed to the mouse&#39;s skull. During training, the headpiece is affixed to a connector that is attached to bars over a cylindrical treadmill in order to stabilize the rodent&#39;s head (Figure 2A). The cylindrical treadmill allows the mouse to rest comfortably, but if the mouse so wishes, also allows it to run or to walk. With the use of this system, mice can be trained with a whisker vibration as the CS and a mild periorbital electrical shock as the US (Figure 1). The US is delivered through wires surgically placed underneath the skin lateral to the eye. The CS is delivered via a comb that is attached to a 2-layer rectangular bending actuator (Figure 2B). The comb and bending actuator are then attached to a magnetic base that is moved to the proper position during training and is adjusted for optimal delivery for each individual animal. The comb is positioned to straddle the selected whiskers. During delivery of the CS, a signal is sent to the bending actuator that displaces the comb and leads to vibration of the whiskers17.
Other stimuli such as a tone or a flash of light have been used as effective conditioned stimuli in mice in the past16,18,19. The reason whisker stimulation is chosen for the CS in this experimental paradigm is the dependence of murine animals on their vibrissae for somatosensory information input during exploration. Whisker stimulation has been shown to be a reliable and effective CS20. Furthermore, given the well-established and organized cortical substrate of the vibrissae system (i.e., the barrel cortex), whisker stimulation as the CS provides an elegant tool for mapping cortical changes and plasticity associated with learning eyeblink conditioning20,21. A head-fixed system allows for the precise stimulation of selected whiskers to compare responses between stimulated neurons and neurons receiving inputs from non-stimulated whiskers. Finally, many strains of mice exhibit age-related hearing loss as relatively young adults22, and eyelid closure during the conditioned blink alters a visual CS (although a visual CS does ameliorate issues with startle responses16). Whisker stimulation is not affected by either of these complications.
Presented here are unique and important modifications upon other head-fixed preparations for eyeblink conditioning, including methods for CS and US delivery, and the acquisition of the eyeblink response. The reliability of this apparatus and the training paradigm in eyeblink conditioning is demonstrated by learning curves from conditioned mice and a relatively flat learning curve from pseudoconditioned control animals (Figure 7A).

<table border="0" cellpadding="0" cellspacing="0" width="1351">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Exervo TeraNova Foam Roller 36&#34; x 6&#34;&#160;</td>
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			<td style="width:181px;">B002ONUM0E</td>
			<td style="width:377px;">For cylinder</td>
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			<td height="21" style="height:21px;width:436px;">Plexiglas</td>
			<td style="width:357px;"></td>
			<td></td>
			<td>Custom-made; 1 cm thick</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Metal Rods (12.7mm diameter)</td>
			<td style="width:357px;"></td>
			<td></td>
			<td>Custom-made</td>
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			<td>&#160;B00F33Q8QO</td>
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			<td height="20" style="height:20px;width:436px;">2-Layer Rectangular Bending Actuator</td>
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			<td>T220-A4-303X&#160;</td>
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			<td height="20" style="height:20px;width:436px;">Threaded rod for magnetic base</td>
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			<td height="20" style="height:20px;width:436px;">0-80 stainless steel machine screw (1 in. long)</td>
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			<td height="40" style="height:40px;width:436px;">Polyimide coated stainless steel wires (2 in. long, .005 in. diameter)</td>
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			<td>For headpiece, EMG and shock wires</td>
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			<td style="width:357px;">Tenma</td>
			<td>72-110</td>
			<td>For the whisker stimulation; rheostat to adjust current to the bending actuator</td>
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			<td height="20" style="height:20px;width:436px;">Amplifier</td>
			<td style="width:357px;">A-M Systems</td>
			<td>1700</td>
			<td>Amplifier for filtering and amplifying EMG signals</td>
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			<td height="20" style="height:20px;width:436px;">WPI A385R stimulus isolator</td>
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			<td>31405</td>
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			<td height="18" style="height:18px;width:436px;">Isothesia (Isoflurane)</td>
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			<td height="20" style="height:20px;width:436px;">Micro Clip</td>
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</table>

whisker-signaled eyeblink classical conditioning in head-fixed mice

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.

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 recordings for each trial were rectified and integrated with a 10 msec time constant. Figure 5 shows the rectified and integrated EMG response traces averaged across all trials for each of 10 sessions for both a conditioned (A) and a pseudoconditioned (B) mouse. The evolution of conditioned responses can be seen in Figure 5A, with responses getting larger closer to the onset of the US. This evolution is not seen in the pseudoconditioned responses in Figure 5B.
The evolution of well-timed, and thus, well-learned conditioned responses can also be seen in Figure 6. Figure 6 shows the histograms of the time to the peak of the response following CS onset. There is an initial peak in the graph between 0 - 150 msec after CS onset. This initial peak can be seen as early as in session 1. With more training sessions, there is the development of a second peak in the graph between 400-500 msec amongst the conditioned animals, indicating the development of better-timed conditioned responses.
Figure 7A shows the averaged percent adaptive conditioned responses recorded from conditioned and pseudoconditioned mice. Adaptive conditioned responses are considered to have significant activity that is present at least 20 msec before US onset (i.e., at least 4 SD greater than the mean activity present 250 msec before CS onset). Over the training sessions, conditioned mice showed gradual learning of the conditioning paradigm by exhibiting more conditioned adaptive responses with each training session so that conditioned mice exhibited significantly more adaptive conditioned responses than pseudoconditioned mice (groups, F(1,15)=20.62, p&#60;.0005; sessions, F(1,9)=9.987, p&#60;.0001; groups *sessions, F(1,9)= 5.977, p&#60;.0001). Note that the pseudoconditioned group typically exhibited pseudo CRs on less than 20% of the trials.
Figure 7B shows the averaged conditioned response area (the area under the curve of the rectified and integrated EMG response) for both conditioned and pseudoconditioned mice over the ten training sessions. Planned comparisons with a repeated measures ANOVA over the last four sessions (i.e., sessions 7-10, once the conditioned group had reached a learning criterion of 60% CR) show a significant main effect of group, indicating that the CR area was larger in the conditioned group than in pseudoconditioned group post learning criterion (groups, F(1,14)=5.733, p&#60;.05; session, F(1,3)=.486, ns; groups*session, F(1,3)=.432, ns).
Figure 7C shows the averaged percent alpha (startle) responses recorded from the conditioned and pseudoconditioned mice. Alpha responses are activity within 50 ms of CS onset that were at least 4SD above the mean baseline activity. Over the training sessions, conditioned and pseudoconditioned mice typically exhibited alpha responses on less than 25% of the trials, with no significant differences between conditioned and pseudoconditioned mice (groups, F(1,15)=2.502, ns). The repeated measures ANOVA did, however, reveal a significant interaction of groups and sessions due to the decrease in pseudoconditioned responses and the maintenance of alpha responses at about 25% for conditioned mice (groups*sessions, F(1,9)=2.074, p&#60;.05). The increase in alpha responses during session 10 is likely due to the short onset latency of the well-developed CR.
Figure 7D shows the averaged percent short latency response recorded from both groups of mice. Short latency responses reflected activity between 50-70 ms following CS onset that was 4SD above the mean baseline activity. A repeated measures ANOVA revealed that conditioned animals exhibited more short latency responses than pseudoconditioned animals over the ten training sessions (groups, F(1,15)=5.377, p&#60;.05; session, F(1,9)=3.920, p&#60;.0005; groups*session, F(1,9)=3.158, p&#60;.005). This may reflect an earlier onset time of the CR, as the conditioned group exhibited larger CRs with each training session.
<img alt="Figure 1" src="/files/ftp_upload/53310/53310fig1.jpg" /> 
Figure 1: The Trace Eyeblink Conditioning Paradigm. In trace eyeblink conditioning, an innocuous conditioned stimulus (CS) is paired with a mildly noxious unconditioned stimulus (US) meant to induce an eyeblink response. The CS and US are separated by a stimulus-free temporal gap, making the paradigm hippocampus-dependent23. In the paradigm described here, a 250 msec long whisker vibration CS is paired with a 100 msec long periorbital shock US. The CS and US are separated by a 250 msec long temporal gap. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53310/53310fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53310/53310fig2.jpg" /> 
Figure 2: The Head-fixed Preparation. A, The head-fixed set-up for eyeblink conditioning with the cylinder, the whisker stimulator, and the connector indicated. B, The whisker stimulator with the two-layer bending actuator and comb indicated. C, The connector with the individual parts indicated: locking screw, the 7-hole strip, the wires soldered to the sockets and epoxy to preserve the wires, the wings converted from a TO-220 style transistor. D, The headpiece with the individual parts indicated: The 7-hole strip, the pins, and the wires soldered into the pins, including the uncoated stainless steel ground wire, and the individual shock and EMG stainless steel wires, with 0.2 cm of polyimide coating stripped off the end. E, The configuration for each hole in the 7-hole strip for both the connector and the headpiece. F, A mouse with a surgically implanted headpiece. G, A mouse is placed on the cylinder, ready to be trained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53310/53310fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53310/53310fig3.jpg" /> 
Figure 3: Example EMG recording. The blue lines delineate the CS presentation (250 msec long). The red lines delineate the US presentation, which shows the artifact that comes from the electrical shock (100 msec). The CS and US are separated by a 250-msec stimulus-free interval. Within this stimulus-free interval is higher amplitude EMG activity (relative to baseline) which delineates a CR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53310/53310fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53310/53310fig4.jpg" /> 
Figure 4: Example EMG and corresponding optical infrared reflection recordings. The EMG recording is just as accurate as the optical infrared reflection sensor in detecting conditioned response onset (blue line), latency to response peak (red line), and unconditioned eyeblink response onset (pink line). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53310/53310fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53310/53310fig5.jpg" /> 
Figure 5: Averaged Eyeblink Traces. Averaged rectified and integrated traces of the eyeblink response for one conditioned mouse (A) and one pseudoconditioned mouse (B). Each trace represents the average response of the mouse for all trials over a single session. The blue section represents the response during the whisker vibration CS presentation. The red section represents the response during the stimulus-free trace interval. In A, the shock US induced an artifact that is present during the dark grey area after the trace interval. B shows the responses to the whisker vibration CS only. Presentation of CS alone and US alone trials was pseudorandomized in pseudoconditioning. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53310/53310fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53310/53310fig6.jpg" /> 
Figure 6: Histograms of the Response Peak Times. Histograms of the time to the response peak for all conditioned (n=8; blue bars) and 8 pseudoconditioned (red bars) animals for all trials across all sessions (data from one pseudoconditioned mouse was excluded to allow a direct comparison of the number of responses between groups). Purple bars indicate where the conditioned and pseudoconditioned animals overlap. Response peak times are calculated as the time of the largest peak of the rectified and integrated EMG recording between CS and US onset. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53310/53310fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53310/53310fig7.jpg" /> 
Figure 7: Response Curves of Conditioned Animals (n=8; blue curves) and pseudoconditioned animals (n=9; red curves). A, Percent Averaged Adaptive Conditioned Responses (&#177; SEM) for conditioned and pseudoconditioned animals over 10 training sessions. B, Averaged area (&#177; SEM) of the CR for conditioned and pseudoconditioned animals over 10 training sessions. C, Percent Averaged Alpha Response (&#177; SEM) for conditioned and pseudoconditioned animals over 10 training sessions. D, Percent Averaged Short Latency Responses (&#177; SEM) for conditioned and pseudoconditioned over 10 sessions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53310/53310fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice at JoVE.com

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

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.

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive ...

whisker-signaled-eyeblink-classical-conditioning-in-head-fixed-mice

Nanyang Technological University

Research

JoVE Journal

Behavior

12.9K Views.  Northwestern University. 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.

Video: Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

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. ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Assessing Activity-based Anorexia in Mice

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions lead to a life-threatening reduction in body weight. However, rodents exposed to only one of these conditions ultimately adapt to re-establish normal body weight. Although increased running coupled with reduction in voluntary food intake appear paradoxical under ABA conditions, ABA behavior is observed across numerous mammalian species. 
The ABA paradigm provides an animal model for anorexia nervosa (AN), an eating disorder with severe dysregulation of appetite-behavior. Subjects are singly housed with free access to a running wheel. Each day, the subject is offered food for a limited amount of time. During the course of the experiment, a subject's body weight decreases from high activity and low caloric intake. The duration of the study varies based on how long food is offered daily, the type of food offered, the strain of mouse, if drugs are being tested, and environmental factors.
A lack of effective pharmacological treatments for AN patients, their low quality of life, high cost of treatment, and their high mortality rate indicate the urgency to further research AN. We provide a basic outline for performing ABA experiments with mice, offering a method to investigate AN-like behavior in order to develop novel therapies. This protocol is optimized for use in Balb/cJ mice, but can easily be manipulated for other strains, providing great flexibility in working with different questions, especially related to genetic factors of ABA.

Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions ...

Classical Short-Delay Eyeblink Conditioning in One-Year-Old Children

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a puff of air to the cornea). Eyeblink conditioning is often used experimentally to detect abnormalities in cerebellar-dependent learning and memory that underlies this type of associative learning. While experiments in adults and older children are relatively simple to administer using commercial equipment, eyeblink conditioning in infants is more challenging due to their poor compliance, which makes correct positioning of the equipment difficult. To achieve conditioning in one-year-old infants, a custom-made or an adapted commercial system can be used to deliver the air puff to the infant's cornea. The main challenge lies in successfully detecting and classifying the behavioral responses. We report that automated blink detection methods are unreliable in this population, and that conditioning experiments should be analyzed using frame-by-frame analysis of supplementary video camera recordings. This method can be applied to study developmental changes in eyeblink conditioning and to examine whether this paradigm can detect children with neurological disorders.

This protocol describes an eyeblink conditioning paradigm appropriate for experiments with one-year-old infants. Commercial or custom-made equipment can be used to deliver the stimuli, and data collection and analysis should be performed on the video recordings.

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...

Operant Conditioning Task to Measure Song Preference in Zebra Finches

An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting opening and indicate their preference for a song by landing on a perch within each chamber. By interrupting the infrared beam from a photoelectric sensor above each perch, the bird activates the playback of a song through a speaker located on each side of the cage. Freely available software is used to trigger the song playback from each perch. To determine the song preference of each animal, her chamber preference is first identified by triggering no song playback when she lands on each perch. This chamber preference is then compared to her song preference. A minimum activity threshold is set to ensure the preference is real. Using this method, we show that paired females prefer the song of their partner. This method was used to understand the contribution of dopamine to the formation and maintenance of song preference.

We describe a technique to evaluate song preference in zebra finches. Females are placed in a two-chambered cage and song preference is measured by the number of times she triggers the playback of one song by landing on a perch within one chamber, compared with triggering a different song in the second chamber. Perch landings are counted using infrared sensors.

An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting ...