Use head-fixation to identify neurons associated with voluntary movements

Use head-fixation to identify neurons associated with voluntary movements

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.

Experimental Procedure

Juxtacellular and multi-unit recordings were taken from the motor cortex of 74 rats who were head-restrained and trained to repeat voluntary forelimbs movements. The juxta-­cellular recording technique was implemented as it provides accurate spike events and morphological features for a cortical or subcortical neuron. The multiunit recording technique is useful for exploring the synaptic connectivity of many neurons simultaneously while remaining blind and unbiased.

Amuza TaskForcer

A multi-­rat task-­training system was developed to simultaneously train up to six adult rats on an operant voluntary forelimb-­movement task. During the operant trials, the trainee rats quickly learned the relationship between the lever and the water reward. This led to the trainee rats casually grabbing the lever with their right forelimb for their reward instead of struggling during the restraining process. 

TaskForcer Lever

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.

Focus on results!

When research animals are stressed or distracted, training can slow and your results are what will suffer variability. Unlike other systems that allow rodents to essentially “run free” while attempting to perform tasks at the same time, Amuza’s TaskForcer eliminates those distractions—and their effects on your data.


*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.

Accurately monitor reward-oriented licking

Accurately monitor reward-oriented licking

Identifying efficient robust methods to monitor licking behavior in rodents is key to
understanding the role of reward-oriented dopaminergic neural pathways in animal behavior. By
monitoring licking behavior, researchers can better understand how rodents gauge the outcome
of a specific reward, their incentive for the reward and how they predict the reward (1).

However, licking microcircuitries in the brain are complex, and incorporate a number of different
neurons controlling different behaviors. A difficult task in recent times has been accurately
identifying the specific microcircuit associated with each specific reward-oriented lick behavior.

The mesolimbic dopamine system is involved in reward-oriented behaviours, and dopamine
antagonism in rodents has been shown to change ingestive behaviors. Pharmacology DAergic
stimulation of the NAc triggers an intense response to obtain a reward, even if a rat has
undergone extinction training (1). While ingestive behaviors include both feeding and drinking,
the exact involvement in water drinking remains unclear.

As mice respond to sensory stimuli by licking for liquid rewards, precise monitoring of licking
during these tasks provides an accessible metric of sensory-motor processing, particularly when
combined with simultaneous neural recordings or microdialysis (2). The precise timing of reward
consumption is critical to understand associations between neural activity and animal behavior.
Therefore using the right detection method as well as a reliable rodent model of dopamine
ensures that licks are monitored reliably (3).

Licking Units – Limitations to be considered

Some of the main challenges when developing and implementing lick detectors during head-restraint
microdialysis or neurophysiological experiments in mice include:

    1.  Electrical contact sensors that trigger food or water feeder to dispense can create
      electrical artifacts that are similar to neural or behavioral amplitudes and time courses,
      which can also interfere with electrophysiology recordings (4).
    2. Temporal characteristics of licking (approx 7Hz) are different from the profile of individual
      licks which are much faster. This is important if trying to determine the onset/offset of
      licks. If lick is being used to send TTL signals to other devices it can be troublesome.
    3. Mice are small, so behavior can be disturbed by equipment that is bulky and obstructs
      animal view.
    4. Head-restraining animals without proper habituation increases cortisol levels and could affect neural recordings / microdialysis. There it is important to ensure adequate training time

Contactless photo-sensors such as infrared detectors overcome these obstacles when
monitoring lick behavior and remove any electrical artifact interference from the set-up.

Fig. 1: Transgenic construction of DSI mice.

Different promoters expressed in each line of transgenic model. Tamoxifen administration used to induce activation of transgenic phenotype.
Dopaminergic synaptic vesicles are prevented from releasing neurotransmitters by v-SNARE cleaving in DSI models.

Case Study
Drinking behavior was analyzed in triple transgenic mice generated with reduced DA release and treated with a D1-like or D2-like DA receptor agonist. Triple transgenic mice were generated to secrete reduced dopamine levels in the striatum and nucleus accumbent compared to control. These triple transgenic mice made fewer licks and fewer lick bursts than control under thirsty conditions. D1 or D2/3 receptor agonists were then administered to identify the influence of dopamine receptors in altered drinking behavior.

New triple transgenic mouse line expected to exhibit partial blockade of synaptic release rather than severely impaired DA secretion seen in other dopamine-depleted mice models. The DSI mouse line enables the study of phenotypes related to DA loss and the role of DAergic neurons and the DA receptors in drinking behavior.

Fig 2: Training of mice to lick for a water reward.

(a) Scheme of the training for licking test. (b) After 2 days of water deprivation, control and DSI mice were trained to lick a water nozzle for a water reward (4 μl/lick) (RM‐ANOVA:genotype, p < .05; time, p < .01). The daily water intake was limited to 1.5 ml per day, and the body weight was maintained at the same level (Ctrl, n = 16; DSI, n = 16). *p < .05 compared to Ctrl mice. Values are shown as the means ± SEMs

Experimental set-up
The apparatus for licking training and data recording includes a water-pumping device and an infrared beam detector system which are controlled by software.

Thirsty mice showed vigorous activity when water was available, and they drank from different angles either in front of or under the water nozzle. This tendency reduced the accuracy of recording. Thus, the researchers utilized an apparatus (TaskForcer, O’Hara) that monitors neural circuits while a mouse is licking. A custom-made head plate was fixed onto a mouse’s skull with dental acrylic to reduce its head movements.

Assessed drinking behavior by analyzing licking microstructure

  • Number of licks and bursts
  • Size of bursts
  • Intraburst lick speed

A burst was defined as continuous licking (>2 licks with <0.4 s between licks).

After 2 days of water deprivation, the mouse was placed inside an acrylic tube and trained to lick for a water reward for 15 mins per day for 7 consecutive days.

Each interruption of the infrared beam counted as one lick, and the mouse was rewarded with one unit of water (4uL of water per lick).

Microdialysis was carried out using equipment supplied by Amuza Inc. to monitor levels of dopamine in the brain of both control and transgenic mice.

Fig 3: Scheme of the rat licking microstructure.

(a) The number of total licks carried out represents the extent of water drinking activity and therefore reflects the general drinking behavior. The number of bursts indicates the activation of responses and thus represents the incentive motivation triggered by reward cues. (b) DSI mice made fewer licks and bursts than the control littermates. The D1 receptor agonist ameliorated the lick number but did not increase the burst number, and the D2 receptor agonist suppressed all the measurement results from the licking test. The D1 agonist A68930 was effective only for DSI mice, but the D1 agonist SKF38393 was effective for both control and DSI mice

Findings suggest that D1 receptor activity impacts drinking and may also contribute to treatment
for illnesses related to DA loss.

DSI mice avoid the infirmity and reduced food and water consumption exhibited by DA-deficient

DSI mice showed impaired motor control when given a challenging rotarod test and made fewer
licks and bursts than control mice.

One D1 receptor agonist increased the number of licks made by thirsty DSI mice.
While another increased the number of licks made by DSI and control mice.

Combine Operant Tasks and Rewards

TaskForcer is the must-have modular system for in-vivo electrophysiology and imaging. Our operant-behavior conditioning system is designed around your priorities. The only animal training system that was created to speed training, simplify configuration, and deliver consistent results to expedite your discoveries.


(1) Kao K‐C, Hisatsune T.: Differential effects of dopamine D1‐like and D2‐like receptor
agonists on water drinking behaviour under thirsty conditions in mice with reduced dopamine
secretion. Eur J Neurosci. 2019;00:1–14. Ht
(2) Williams, B., Speed, A., Haider, B: A novel device for real-time measurement and
manipulation of licking behavior in head-fixed mice (2018)
(4) Hayar, A., Bryant, J.L., Boughter, J.D., Heck D.H.: A low-cost solution to measure mouse
licking in an electrophysiological setup with a standard analog-to-digital converter (2008)

Modular Maze System, the Free Maze Setup

Modular Maze System, the Free Maze Setup

Modular Maze System, The Free Maze Setup

The Free Maze is quite easy to configure, you start by screwing all of the fixed stands into place on the breadboard floor.

The orientation and position can be easily changed to give you different designs, simply slide the pegs at the bottom of each stand into a hole on the floor and then use the round black screw on an empty hole to secure it into place.

Next, you will attach each corridor unit. For the T-maze set-up, the center T corridors will divide the maze in 2, the straight corridors make up the length of each side of the maze, and then the end left and right corridors will sit under the pellet dispensers.

Finally, you will go ahead and attach the pellet dispenser units. In this video, they are placed at opposite ends of the T-maze


O’Hara Behavioral Testing Equipment

O’Hara Behavioral Testing Equipment

Built out of a desire to help standardize behavioral neuroscience research

One of our brands O’Hara, a manufacturing company based out of Japan, has been developing and manufacturing equipment for behavioral experiments for over 40 years. O’Hara got its start by designing and manufacturing equipment for pharmaceutical companies that were doing efficacy testing on various therapeutic drugs. They quickly realized that there was no method of standardization across behavioral studies from different labs, research institutes, and pharmaceutical companies, which they felt would have detrimental consequences on data integrity. With this issue at the forefront of their minds, O’Hara began developing automated behavior tests to improve the reliability of behavioral data. This newfound focus was born out of a desire to help improve the reproducibility crisis that has been plaguing Neuroscience research for several years now – That is the inability to replicate scientific studies across experiments and research institutions.

What is the reproducibility crisis?

Reproducibility or replicability from a research standpoint is the idea that a given set of experimental findings should be able to be replicated following the same procedures. The inability to replicate basic scientific findings across research institutions and even across experiments is a major issue plaguing Neuroscience research today and is particularly prevalent in animal studies. This is not hard to believe given that the use of animals themselves provides inherent variability, even when all other factors are controlled for. To read more about this issue and what we are doing to try and alleviate the problem click here.

Ohara offers a variety of automated systems to help you standardize the data collection process

Don’t see what you are looking for?

O’Hara is committed to making customizable solutions to fit the researcher’s specific needs, and therefore our products can be tailored to better fit your needs. In fact, two of our products, the Free Maze and the Self head-Restraining Platform were created out of collaborations between research labs and O’Hara.

Learn more about the background behind the making of the Free Maze here.

Learn more about the motivation behind the Self Head-Restraining Platform here.

For more detailed information about our automated behavioral solutions connect with us today.

Precise Touch Operant Training

Precise Touch Operant Training

Precise Touch Operant Training

Today I’m going to demonstrate the unique features of our touch panel operant chamber system.

What is unique about our chambers is that they contain infrared sensors located at the top of the touchscreen itself that improve the accuracy of touch responses from small rodents.

Unlike the touch screen technology that most of our smartphones and computer screens use, called projective capacitance, the infrared sensors inside the touch screen improve accuracy by eliminating the need for a minimum force required to generate a response. This means that even nose poke’s from mice will register a response.

In addition to improved sensitivity, our touch panel was designed in a trapezoidal shape instead of a square, making it easier for the animal to focus on the screen ahead.

Our touch panel chambers are compatible with in vivo electrophysiology and optogenetics techniques, as well as with miniature head-mounted microscopes.

Chambers can be purchased singly or in a package of four for a more cost-effective option. In addition, the chambers come with our Operant TaskStudio software package, an extremely user-friendly software platform that enables customers to design and execute their own tasks or choose from a variety of pre-programmed tasks.

Check out more about our software in our next video. To learn more about our Touch Panel operant system please check out our product website or connect with an expert.


Obtain accurate fluid intake measurements

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The Drinko Measurer is one of our most simple products, but an extremely useful one for gathering precise measurements of liquid consumption in animal studies.

What makes the Drinko Measurer unique is that it contains double ball bearings inside the nozzle of the sipper, preventing leakage of excess liquid. Furthermore, when force from a mouse or rat is applied to the tip of the sipper tube, a single drop of solution is applied.

Each tube also contains a clip that attaches to the sipper tube to the cage to stabilize the bottle to the cage. This prevents it from being knocked out of place by the mouse or rat. This clip can also be adjusted so you can adjust the length of the nozzle within the cage.

Additionally, all parts are autoclavable for easy cleaning.

The Drinko Measurer comes with several different sipper tube lengths 2.5, 3.5 and 4.5 inch as well as two different bottle sizes, 10 and 15ml. We also have new 15ml bottles with ml markings making it easier to visualize liquid consumption. However, we recommend weighing the bottles for the best results.

Now I’m going to tell you about several applications we recommend for the Drinko Measurer. The first is for drug delivery experiments.
For drugs that can be administered orally and easily mixed into a solvent, simply use the Drinko Measurer as a means for drug delivery rather than having to inject your experimental animals.
This is useful for drug dosing experiments and toxicology studies. Also, by combining two units per cage, you can measure drug-seeking preference with drugs of abuse.

The second application is for conditioned taste preference. Or conditioned taste aversion
Easily Measure preference or avoidance of a liquid by combining two Drinko Measurer systems and measuring liquid consumption.

Finally, we do carry 40 mL bottles which are useful for larger animals or for extended access studies. 

You can purchase units easily by navigating to our shop.