Rodent Head-Fixation System

• Introduction
• Apparatus
• Data
• Protocol
• Literature
• Strengths
• References

Introduction

Head fixation serves as a fundamental procedure in neurological research. Its primary role is to immobilize subjects, thereby facilitating optical assessment of neuronal circuits and reducing background noise and motion artifacts. The head fixation system limits the range of behavioral movements, crucial for studying learning behaviors related to reward and specific motor skills such as eye movements, lever pulling, licking, and engaging in virtual reality scenarios (Weaver et al., 2023).

Moreover, head fixation devices are extensively utilized in neuroscience laboratories for recording neuronal activity through techniques such as calcium imaging and electrophysiology.

The process of head fixation is essential as it influences head and eye movements in response to visual stimuli. Beyond motor reflex studies, head fixation devices are instrumental in training mice for operant conditioning tasks and facilitating differentiation between auditory, visual, and olfactory stimuli.

To implement head fixation, the head bar must be securely attached to the animal's head, followed by immobilization of the head bar and the subject's head using an external apparatus. A head hat is also used to protect hardware mounted on the skull.

Apparatus and Equipment

The head fixation system features a metal restrainer platform and removeable lid, allowing for easy movement of the subject. This restrainer ensures superior stability and comfort for the subject throughout the experiment. Thumbscrews allow easy capture and release of the animal.

The default aluminum headplate has a 6 mm x 6 mm skull window and is fixed with M2.5 cross countersunk screws. 

Customizations include material composition and dimensions, please enquire

Mazeengineers provide an ergonomically designed head fixation system tailored to research needs.

Data Analysis

The following can be studied using the Rodent Head-Fixation apparatus:

• Drug and anesthesia effects
• Rodent psychophysics
• Rodent fine motor skills such as facial function eg. blinking
• Motor skills such as reflex adaptions and actions
• Neural and biomechanical force production
• Neuroimaging techniques such as Optogenetics
• Electrophysiology
• Operant conditioning

Protocol

1. Deeply anesthetize the mice using 2% isoflurane and mount the subject on the stereotaxic frame.
2. Place the mice on a heating pad and cover its eyes in a thin layer of petroleum jelly.
3. Clean the subject’s scalp with 70% ethanol and betadine and inject 50ꭒl 0.5% Marcaine under the scalp for topical anesthesia. In addition, inject an anti-inflammatory drug like ketofen subcutaneously and buprenorphine intraperitoneally.
4. Following this, remove approximately 1cm2 flap of skin from the dorsal skull with a single cut and clean the remaining gelatinous periosteum with small scissors. Then clean and dry the skull using a sterile cotton swab.
5. Expose the skull by scrapping off the bone with a scalpel and cover the exposed skull with a layer of cyanoacrylate glue.
6. Place the head bar directly into the wet glue. Use dental cement to cover the glue and secure the head bars in place.

1. Place the wings of the head bar into the notches in a stainless steel holder using a pair of thumbscrews and clamps.
2. Now, insert the mouse body into the acrylic body tube such that the mouse’s head extends out and its front paws grip the tube’s edge after head fixation.
3. After that, attach the holder along with the body tube to a caddy. The head bar should be 30mm above the body tube’s bottom. Fix the caddy into the behavior box. Always remember that a head-fixed mouse should crouch in the body tube in a natural position.
4. Following head fixation, the mice can be subjected to various experiments by using accessories like lickport to study the neurobiology of rodent behavior.

1. Deeply anesthetize the mice using 2% isoflurane and mount the subject on the stereotaxic frame.
2. Place the mice on a heating pad and cover its eyes in a thin layer of petroleum jelly.
3. Clean the subject’s scalp with 70% ethanol and betadine and inject 50ꭒl 0.5% Marcaine under the scalp for topical anesthesia. In addition, inject an anti-inflammatory drug like ketofen subcutaneously and buprenorphine intraperitoneally.
4. Following this, remove approximately 1cm2 flap of skin from the dorsal skull with a single cut and clean the remaining gelatinous periosteum with small scissors. Then clean and dry the skull using a sterile cotton swab.
5. Expose the skull by scrapping off the bone with a scalpel and cover the exposed skull with a layer of cyanoacrylate glue.
6. Place the head bar directly into the wet glue. Use dental cement to cover the glue and secure the head bars in place.

1. Place the wings of the head bar into the notches in a stainless steel holder using a pair of thumbscrews and clamps.
2. Now, insert the mouse body into the acrylic body tube such that the mouse’s head extends out and its front paws grip the tube’s edge after head fixation.
3. After that, attach the holder along with the body tube to a caddy. The head bar should be 30mm above the body tube’s bottom. Fix the caddy into the behavior box. Always remember that a head-fixed mouse should crouch in the body tube in a natural position.
4. Following head fixation, the mice can be subjected to various experiments by using accessories like lickport to study the neurobiology of rodent behavior.

Literature Review

Hughes et al. (2020) tried studying the subtle movement and postural adjustments that animals experience during head fixation. The experimenters utilized a novel mouse head fixation system equipped with five orthogonal force sensors to study in vivo electrophysiology of the mouse brain during behavioral tasks like licking.

They housed the mice in groups of 3-4 mice per cage following a 12:12 light cycle and conducted the experiments in the light phase. The subjects were deprived of water and allowed free access to water for about 2h after the experimental sessions. The scientists attached the mouse perch and head fixation apparatus to a base plate made of steel and elevated this assembly to accommodate the reward delivery apparatus. The load cells were also added to the experimental setup. Following this, the researchers anesthetized the mice with 2-3% isoflurane and performed craniotomy using a stereotaxic frame. During the surgery, they implanted 16 electrodes in a 4×4 configuration using thumbscrews and secured them using dental acrylic. Then, they also inserted a metal bar and cemented it using dental acrylic.

Ultimately, the experimenters trained the mice for a Pavlovian conditioning task and observed the licking behavior. They concluded that licking-related oscillations revealed postural alterations among the subjects.

Hughes et al. (2020) tried studying the subtle movement and postural adjustments that animals experience during head fixation. The experimenters utilized a novel mouse head fixation system equipped with five orthogonal force sensors to study in vivo electrophysiology of the mouse brain during behavioral tasks like licking.

They housed the mice in groups of 3-4 mice per cage following a 12:12 light cycle and conducted the experiments in the light phase. The subjects were deprived of water and allowed free access to water for about 2h after the experimental sessions. The scientists attached the mouse perch and head fixation apparatus to a base plate made of steel and elevated this assembly to accommodate the reward delivery apparatus. The load cells were also added to the experimental setup. Following this, the researchers anesthetized the mice with 2-3% isoflurane and performed craniotomy using a stereotaxic frame. During the surgery, they implanted 16 electrodes in a 4×4 configuration using thumbscrews and secured them using dental acrylic. Then, they also inserted a metal bar and cemented it using dental acrylic.

Ultimately, the experimenters trained the mice for a Pavlovian conditioning task and observed the licking behavior. They concluded that licking-related oscillations revealed postural alterations among the subjects.

Strengths & Limitations

•  The animals chosen for head-cap implantation using a head fixation system should be optimally 12 weeks of age. By this time, the females are fully grown up whereas males can grow for a longer time.
• Make sure that the animal becomes acquainted with the researcher before conducting the behavioral task. For this, simply place your hand in the cage and let the animal explore it.
• If you’re using a cream for depilation, make sure to properly rinse it afterwards.
•  During stereotaxic surgery using a head fixation device, use a heating pad to maintain the animal’s body temperature at 37oC (Schwarz et al., 2010).
•  Try employing single-housing mice to prevent the damage caused to implants by cages.

The head fixation system is a highly effective tool featuring an ergonomic design utilized in neurobiology laboratories to secure murine heads. A primary advantage of these devices is their ability to facilitate precise monitoring of neuronal activity in mice, thereby minimizing background noise and motion artifacts. The mechanical stability provided by head fixation creates optimal conditions for visualizing neural activity in awake animals. Additionally, these devices offer researchers greater experimental control and ensure electrophysiological neural signals remain unaffected even without task-specific training (Schwarz et al., 2010). Modern head fixation systems also occupy minimal space on the subject’s head.

However, there are several drawbacks to consider. For example, acclimating a head-fixed behaving rat to the experimental setup can be time-consuming. Furthermore, the system restricts the animal’s ability to perform extensive whole-body movements. Lastly, head bars wider than the subject’s body may require extended time for effective head fixation.

•  The animals chosen for head-cap implantation using a head fixation system should be optimally 12 weeks of age. By this time, the females are fully grown up whereas males can grow for a longer time.
• Make sure that the animal becomes acquainted with the researcher before conducting the behavioral task. For this, simply place your hand in the cage and let the animal explore it.
• If you’re using a cream for depilation, make sure to properly rinse it afterwards.
•  During stereotaxic surgery using a head fixation device, use a heating pad to maintain the animal’s body temperature at 37oC (Schwarz et al., 2010).
•  Try employing single-housing mice to prevent the damage caused to implants by cages.

The head fixation system is a highly effective tool featuring an ergonomic design utilized in neurobiology laboratories to secure murine heads. A primary advantage of these devices is their ability to facilitate precise monitoring of neuronal activity in mice, thereby minimizing background noise and motion artifacts. The mechanical stability provided by head fixation creates optimal conditions for visualizing neural activity in awake animals. Additionally, these devices offer researchers greater experimental control and ensure electrophysiological neural signals remain unaffected even without task-specific training (Schwarz et al., 2010). Modern head fixation systems also occupy minimal space on the subject's head.

However, there are several drawbacks to consider. For example, acclimating a head-fixed behaving rat to the experimental setup can be time-consuming. Furthermore, the system restricts the animal's ability to perform extensive whole-body movements. Lastly, head bars wider than the subject's body may require extended time for effective head fixation.

Summary

A head fixation system is employed in neurobiology laboratories to affix mouse heads during several behavioral paradigms.

• A head fixation system comprises a head restrainer pre-mounted on a metallic breadboard.
• It also contains thumbscrews which can be connected to titanium head bars (purchased separately), and a clear acrylic tube to hold the animal during the surgery.
• Head fixation devices are commonly used for studying anesthesia effects, facial function, neuroimaging, reflex adaptation, operant conditioning, and reflexes such as eye blinking in mice.

References

Guo, Z. V., Hires, S. A., Li, N., O’Connor, D. H., Komiyama, T., Ophir, E., … & Svoboda, K. (2014). Procedures for Behavioral Experiments in Head-Fixed Mice. PLoS ONE, 9(2).

Hughes, R. N., Bakhurin, K. I., Barter, J. W., Zhang, J., & Yin, H. H. (2020). A head-fixation system for continuous monitoring of force generated during behavior. Frontiers in integrative neuroscience, 14, 11.

Schwarz, C., Hentschke, H., Butovas, S., Haiss, F., Stüttgen, M. C., Gerdjikov, T. V., … & Waiblinger, C. (2010). The head-fixed behaving rat—procedures and pitfalls. Somatosensory & motor research, 27(4), 131-148.

Weaver, I. A., Yousefzadeh, S. A., & Tadross, M. R. (2023). An open-source head-fixation and implant-protection system for mice. HardwareX, 13, e00391.