1. Head-Fixation in Neuroscience Research

Head-fixation is widely used in the field of behavioral neuroscience in awake behaving animals to better understand the cortical involvement in animal behaviors. Our understanding of topics such as associative learning, sensory perception, navigation and motor control have been greatly improved by head-fixed experimental preparations. The restraint and motion minimization of the animal’s head minimizes noise and motion artifacts often seen in freely moving studies, which allows for stimulus control studies, perturbation experiments, neural recordings and in vivo cellular imaging. It also simplifies the experimental set-up design and data analysis, allowing for chronic and long-duration studies as many systems facilitate accurate repeated alignment between the animal’s head and the restraining system.

Most recently, head-fixation has been used alongside advanced techniques such as high-density electrophysiology recordings and two-photon imaging to investigate neural circuits in vivo that would not be possible to study in freely moving subjects.

2. Limitations to Most Head-Fixation Systems

However, there are a number of limitations that need to be considered when carrying out large-scale monitoring of neuronal activity under head-fixed operant conditions.

• Many models of head-fixation assume that head movements don’t occur when the animal is restrained. Some subtle movements of the animal’s head that cannot be easily observed or taken into consideration when doing data analysis can result in noise or artifact production. This can lead to significant confounding variables, with a misinterpretation of neural activity associated with animal behavior. Choosing the correct head-fixation instrumentation reduces the risk of confounding variables associated with involuntary head movements.
• Measuring motor responses in rats or mice by monitoring whisker movements and licking behavior have been used in conditional behavioral tasks in the past. However, these motor movements are driven by a pathway in the brain involving automatic repetition that involves the brainstem. Identifying a reliable, measurable movement under operant learning conditions is imperative to accurately associate cortical activity with behavior.

3. Skilled Motor Movements in Operant Conditioning

Skilled motor movements are a good measure of operant learning, as rats and mice are naturally able to perform voluntary skilled motor movements using their forelimbs. Skilled movements are intentional isolated movements with specific parts of the body, which require the recruitment of different neural pathways that contain different subtypes of neurons firing with synchronicity. A number of operant learning paradigms can be studied by measuring forelimb movement response under head-restraint conditions alongside high-density electrophysiology recordings or two-photon imaging. It is important to consider the type of restraining system to use when studying voluntary forelimb movement in rodents.

4. Case Study – Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements

Using the TaskForcer (O’Hara) restraining system, The Isomura* group at the University of Tokyo uncovered functional diversity of pyramidal cells and the uniformity activation of fast-­spiking interneurons across all cortical layers in the expression of trained rodent voluntary movement. Furthermore, they identified a pattern of excitatory synaptic interactions among neighboring neurons that play different roles in self-initiated voluntary forelimb movement.

All 74 rats were successfully able to perform the operant motor task in just 8 days of training. Post training, the rats were transferred to a recording room where they were able to perform the same casual operant motor task during juxtacellular and multiunit recordings. Several distinct  patterns of neuronal firing at an electrode site in relation to the forelimb-­movement task were revealed during these multi-unit recordings.

Resources

*Isomura Y, Harukuni R, Takekawa T, Aizawa H, Fukai T. Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements. Nat Neurosci. 2009 Dec;12(12):1586-93. doi: 10.1038/nn.2431. Epub 2009 Nov 8. PMID: 19898469.

Cranial window implants in head-fixed mice offer stable optical access to large areas of the cortex over extended periods of time. Window preparations can be combined with viral preparations (or in genetically modified mice) to monitor, map or manipulate neuronal activity (eg. using optogenetics) in awake behaving animals. This makes them extremely useful for studying relationships between neuronal activity and behavior.

A major roadblock with the cranial window and optical imaging approach.

The main difficulty with the cranial window and optical imaging approach is the alignment of the mouse or other experimental subjects under the microscope. Due to the curvature of the subject animal’s skull, cranial windows are almost always angled. However, for the most precise optical access, windows should be aligned parallel to the imaging objective. Even minor changes to the window angle can offset neuropil in the axial direction under the microscope and sacrifice data collection. This issue becomes compounded when you repeat experiments over time. Minor changes in the window angle across sessions can distort or change the location of neuropil within the same imaging region, making it challenging to draw significant conclusions from your data.

How can you precisely align the mouse under the microscope each time?

One of AMUZA’s tools, the Imaging Adapter Base Mount on the TaskForcer, overcomes this challenge.

While the mount was made for the TaskForcer unit, it can be made to fit any behavioral rig and enables a more precise fit of your behavioral setup under the microscope.

The Imaging Adapter Base mount can be rotated in X, Y and Z directions to change the angle of your behavioral setup so that you can precisely align the head of the mouse underneath the microscope.

The base mount also contains mm markings, allowing you to get the exact same fit each time you place your behavioral rig under the microscope. This greatly minimizes the chance that observable changes to neuropil are due from distortion of the imaging location under the microscope and greatly improves the quality of your data.

If you are imaging over a wide field of view, the Imaging Adapter Base Mount can be readjusted to align each separate imaging location parallel with your objective. This ensures a consistent level of accuracy across each imaging location.

Each Imaging Adapter Base Mount is custom built to fit your microscope to ensure that you are getting the best product tailored to your specific needs.

In an era of Neuroscience where reproducibility of data is crucial, the Imaging Adapter Base Mount and TaskForcer unit offer stable precision for the most accurate results. You can learn more about this product, and the TaskForcer on our TaskForcer webpage.

Tanaka and colleagues in Dr. Matsuzaki’s lab at the University of Tokyo have been researching the role of thalamocortical axonal activity in motor learning using the TaskForcer. 

Brain regions involved in voluntary movement

The thalamus is a central hub through which neuronal signals are transmitted through the cortex and other subcortical structures including the basal ganglia, the pons, and the cerebellum.

Together, these structures are involved in controlling voluntary movements like manual skills. In animals, manual skills are learned and refined through repetitive motor learning, which instigates neuronal plasticity in the brain structures involved in these processes.

Measuring axonal activity in vivo

Using two-photon calcium imaging of GCaMP expressing thalamocortical axons in the mouse motor cortex in combination with the TaskForcer restraint operant chamber, Tanaka, et al., ascertained the role of thalamocortical axonal activity in skilled motor learning.

The TaskForcer operant chamber fits under the 2P microscope, enabling precise neural imaging during operant training. The task used was a self-initiated lever-pull task, where mice were trained to pull a lever in order to receive a water reward.

By recording calcium activity of GCaMP expressing thalamocortical axons in the motor cortex during learning, they were able to track the temporal dynamics of thalamocortical activity associated with each stage of the learning process.

Linking neuronal activity to coordinated movements

The authors found that thalamocortical activity was time-locked to both initiation and execution of the lever pull task and that this activity stabilized over time after the initial learning. As proof of concept to verify the thalamus’ role in motor learning, when the authors lesioned the thalamus, lever pull behavior significantly decreased. These results indicated that thalamocortical axonal activity is necessary for motor skill learning, and is more involved during the initial stages of motor skill learning.

Upon my return back to Tokyo, I had one final visit with Dr. Isomura at Tokyo Medical and Dental University. He originally developed the TaskForcer for rats with O’Hara over 8 years ago!

Dr. Isomura’s research focuses on understanding information processing in Motor Cortex during motor skill learning. To do this, he performs in vivo whole-cell patch clamp recordings in Motor Cortex as animals learn the lever pull task that was specifically designed for the TaskForcer.

What makes simultaneous neural recording during operant behaviors possible with the TaskForcer is the unique spout-lever. This was specially designed by Dr. Isomura and O’Hara such that the reward (liquid from the spout) and operandum (lever) are combined into one. In this way, the animal can still obtain a reward for pulling the lever even while its body is restrained, allowing for operant learning during simultaneous neurophysiological recording.

Dr. Isomura explains, “Since the animals must learn to perform the lever pull task while under head fixation, we wanted to make sure that the animal could access the reward with minimal head movement, but still be motivated to perform the task.”

Isomura also explains, “We were surprised that rats started pulling the lever the very first day that we put them in the chamber. The lever pull task is very robust. We don’t see animal attrition from failure of animals to learn the task.”

Me with Dr. Takahashi at Doshisha University

“With the TaskForcer, we can reliably get extremely precise single unit recordings during motor behaviors which allows us to examine causal links between neural activity and behavior in great detail.” – Dr. Isomura