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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<li>Ward, R. L., Flores, L. C., Disterhoft, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infragranular+barrel+cortex+activity+is+enhanced+with+learning%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">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 Sie...

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).

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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 r...

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

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

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

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

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

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

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

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 ...

A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and ...

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Neuroscience

Behavioral Training Procedures for Head-fixed Virtual Reality in Mice

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays to be performed in head-fixed mice. This enables precise behavioral recordings while incorporating various neurophysiological techniques that require head-fixation to minimize movement-related signal noise during neural recordings. However, despite the growing use of VR, there is little published data on the detailed methodology of how to implement it. In this study, a training protocol is developed whereby male and female C57B16/J mice are trained to run down a virtual linear corridor, the length of which is increased from 1-3 m over multiple training sessions. Building upon this foundation, this study investigated the feasibility of mice performing complex behaviors within VR using a Y-maze paradigm. The task required navigating to the arm with black walls from the choice point in the Y-maze. After reaching a criterion of two consecutive days equal to or greater than 70% correct, the mice progressed to increasingly difficult sensory discrimination. The findings provide important details on the methodologies useful for the successful training of mice in VR and demonstrate that mice exhibit learning capabilities in navigating the Y-maze. The methodology presented not only offers insights into training duration in VR-based assays but also underscores the potential for probing intricate behaviors in mice, opening avenues for more comprehensive neuroscience investigations.

The article describes the experimental procedures for the commonly used linear track virtual reality (VR) paradigm in mice as well as determining the feasibility of running complex VR tasks by testing a Y-shaped signal discrimination task.

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays ...

Real-Time Mouse Eye Squint Detection for Migraine Pain Tracking Using DeepLabCut

An Automated Squint Method for Time-syncing Behavior and Brain Dynamics in Mouse Pain Studies

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head pain, as in disorders such as migraine. Eye squint has emerged as a continuous variable metric that can be measured over time and is effective for predicting pain states in such assays. This paper provides a protocol for the use of DeepLabCut (DLC) to automate and quantify eye squint (Euclidean distance between eyelids) in restrained mice with freely rotating head motions. This protocol enables unbiased quantification of eye squint to be paired with and compared directly against mechanistic measures such as neurophysiology. We provide an assessment of AI training parameters necessary for achieving success as defined by discriminating squint and non-squint periods. We demonstrate an ability to reliably track and differentiate squint in a CGRP-induced migraine-like phenotype at a sub second resolution.

Author Spotlight: Deciphering Electrical Networks Behind Complex Brain Activities and Disorders

This protocol provides a method for tracking automated eye squint in rodents over time in a manner compatible with time-locking to neurophysiological measures. This protocol is expected to be useful to researchers studying mechanisms of pain disorders such as migraine.

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head ...

Assessing Rodent Empathy: Observational Fear and Vicarious Freezing Method

Observational Fear as a Model of Affective Empathy in Mice

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows individuals to respond to the emotional states of others, promoting prosocial behaviors and social bonding. Observational fear is a fundamental aspect of affective empathy, where an observer witnesses a demonstrator undergoing aversive experiences and subsequently exhibits fear behaviors. This socially induced vicarious freezing response in observers, known as emotional contagion or affect sharing, is regarded as an indicator of empathy-like traits in rodents. Here, we present a protocol that delineates the assessment of vicarious freezing responses in observers exposed to conspecific demonstrators receiving aversive foot shocks. The utilization of observational fear assays in rodents has become a widely adopted method for studying the neural mechanisms underlying affective empathy. Given the universality of observational fear across mammals, this methodology further contributes to advancing our understanding of the neural substrates of empathy in humans.

The observational fear paradigm assesses vicarious freezing in rodents as a model for affective empathy. This procedure entails exposing observer rodents to conspecific demonstrators receiving aversive foot shocks to elicit empathic freezing responses. By employing observational fear assays, researchers can investigate the neural mechanisms underlying affective empathy.

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...