3 Key Factors Influencing In-Vitro Optogenetics Experiment Design

3 Key Factors Influencing In-Vitro Optogenetics Experiment Design

Introduction

Optogenetics is a powerful technique that enables researchers to manipulate cellular activity using light. While in-vivo optogenetics focuses on studying neural circuits in living animals, in-vitro optogenetics allows for the investigation of cellular responses and signaling pathways in controlled laboratory settings. When designing in-vitro optogenetics experiments, several key factors come into play.

Opsin Sensitivity

Opsins, the light-sensitive proteins used in optogenetics, are one of the key factors to consider for in-vitro optogenetics research. Opsins are a class of photoreceptor proteins that have a selective sensitivity to various wavelengths of light. Opsins function by changing their molecular structure as a response to receiving specific wavelengths of light. The change in structure results in activation of the G protein, also known as guanine nucleotide-binding proteins, causing a signaling cascade and a physiological response in the target sample. (Shichida and Matsuyama, 2009). 

Different opsins exhibit varying sensitivities to light across the spectrum. For example, channelrhodopsin-2 (ChR2), a commonly used algal protein, is maximally activated by blue and violet light. Similarly, opsins with sensitivities to other wavelengths, such as green, red, or even ultraviolet (UV) light, can be utilized with in-vivo and in-vitro optogenetics experiments to selectively modulate cellular responses and investigate specific signaling pathways. 

The choice of opsins and the corresponding wavelengths used in in-vitro optogenetics experiments depend on your individual objectives and the specific cellular responses under investigation. Understanding opsin’s sensitivity to light is key to successful in-vitro optogenetics experiments and unlocking the mechanisms underlying cellular activity and signaling pathways.

Tissue Penetration

Tissue penetration is an important factor when deciding which wavelength of light to use for your in-vitro optogenetics experiment. Different wavelengths of light exhibit varying levels of tissue penetration. This can unlock the potential to target specific cell types or regions of interest.  

The exact penetration depth can be influenced by factors such as scattering, absorption, and the specific cell types or structures being targeted. Generally, shorter wavelengths, such as ultraviolet and blue light, have lower tissue penetration, meaning they can only reach superficial layers of tissue. On the other hand, longer wavelengths, such as red and infrared light, have greater tissue penetration and can reach deeper layers of tissue.

Here are some examples of different wavelengths and common opsins utilized with each:

Wavelength Penetration
 Light Color  Wavelength  Common Opsin  Description
Ultra-violet 365 nm OptoSTIM1 Ultraviolet light at 365 nm has limited tissue penetration. It is primarily used for the precise excitation of UV-sensitive opsins, providing control over cellular activity in superficial layers of tissue.
Violet 405 nm, 420 nm OptoSTIM1 Violet light at 405 nm and 420 nm penetrates moderately into tissue. It enables precise control over cellular activity in both superficial and deeper layers.
Blue 450 nm Channelrhodopsin-2 (ChR2) Blue light at 450 nm and 470 nm exhibits moderate tissue penetration. It is commonly employed to activate channelrhodopsin-2 (ChR2), inducing depolarization and increased neural activity in both superficial and deeper regions.
Green 525 nm Green-Activated Protein (GAP) Green light at 525 nm penetrates moderately into tissue. It offers a versatile option for activating and inhibiting various opsins, enabling control over neural circuits in both superficial and deeper tissue layers.
Yellow 590 nm Halorhodopsin (eNpHR3.0) Yellow light at 590 nm exhibits moderate tissue penetration. It is commonly used to inhibit neuronal activity using opsins like halorhodopsin, facilitating neural suppression in both superficial and deeper tissue regions.
Red 630 nm, 660 nm, 740 nm ReaChR Red light at 630 nm, 660 nm, and 740 nm provides deeper tissue penetration compared to shorter wavelengths. It enables the activation or inhibition of neurons in deep brain regions due to its effective tissue penetration.
Infra-red 940 nm Jaws Infrared light at 940 nm possesses the deepest tissue penetration among the mentioned wavelengths. It finds applications in stimulating neurons in thick brain slices or reaching deeper brain regions.

 

In-Vitro LED Array

Ready to enhance your in-vitro optogenetics research? Discover the power of Amuza’s LED Array, designed for precise control and manipulation of cellular activity. Visit our website to explore our range of products and take your experiments to the next level.

 

Phototoxicity

Phototoxicity is an important concern with in-vitro optogenetics experiments due to the potential damage it can cause to cells. In 2018, Marvek et al. demonstrated blue-light decreases overall neuronal cell viability and can modify neuronal morphology. However, possible solutions have been proposed within the past few years to address causes of the decreased cell viability as a result of blue light. 

One approach to increasing cell viability during in-vitro research is decreasing blue-light fluorescence intensity. In 2019, Duke et al. observed that increasing blue-light fluorescence intensity was detrimental to cell viability. This was measured by exposing cells to various duty cycles and measuring the impact on cell viability. Duke defies a duty cycle as “light-on-time / total-time x 100.” As the light-on-time in each duty cycle increases, cell viability after 8 hours of exposure decreases. This was measured at duty cycles of 1.67%, 3.33%, and 6.67%.

Different opsins exhibit varying sensitivities to light across the spectrum. For example, channelrhodopsin-2 (ChR2), a commonly used algal protein, is maximally activated by blue and violet light. Similarly, opsins with sensitivities to other wavelengths, such as green, red, or even ultraviolet (UV) light, can be utilized with in-vivo and in-vitro optogenetics experiments to selectively modulate cellular responses and investigate specific signaling pathways. 

The choice of opsins and the corresponding wavelengths used in in-vitro optogenetics experiments depend on your individual objectives and the specific cellular responses under investigation. Understanding opsin’s sensitivity to light is key to successful in-vitro optogenetics experiments and unlocking the mechanisms underlying cellular activity and signaling pathways.

Recommended Case Studies

In the following case studies, a few example applications of some specific wavelengths of light in in-vitro optogenetics research will be highlighted, showcasing the potential of this technique in advancing our understanding of cellular behavior and contributing to the development of targeted interventions in various fields.

Blue Light (470 nm)

In Stierschneider’s publication, blue light was utilized as a tool for in-vitro optogenetics to activate the Toll-like receptor 4 (TLR4) signaling pathway and the NF-κB-Gluc reporter system in human pancreatic cells.

By exposing the cells to blue light, the researchers were able to precisely control the activation of these pathways, allowing for a detailed investigation of their dynamics and regulatory mechanisms in a controlled laboratory setting.

Yellow Light (590 nm)

In 2021, Amitrano et al. used in-vitro optogenetics emitting a yellow light to show that there was an increase in overall mitochondrial function during both human and mouse CD8+ T cell activation. The Amuza LED array (590 nm) was used to illuminate HEK293T cells and CD8+ T cells with the ATP assay.

Blue and Yellow Light in One System (470 nm and 590 nm)

Catanese et al. utilizes a combination of transcriptomics, proteomics, optogenetics, and pharmacological approaches, revealing the accumulation of aberrant aggresomes, reduced synaptic gene expression, loss of synaptic contacts, and dynamic MAL activation of the transcription factor CREB in ALS-related motoneurons. 

The observed pathological features of ALS-related motoneurons included the accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts, and dynamic MAL activation of the transcription factor CREB.

Conclusion

The 3 key factors to consider during in-vivo research are opsin sensitivity, tissue penetration, and phototoxicity. By selecting the appropriate opsins and wavelengths of light, researchers are able to investigate cellular responses and signaling pathways in their target tissue while keeping phototoxicity to a minimum.

Through the recommended case studies, we have witnessed the ability of specific light wavelengths in studying signaling pathways, mitochondrial function, and disease-related features. Researchers are encouraged to explore tools like the Amuza LED Array to further enhance their experiments.

Looking for information discussing In-Vivo Optogenetics?

In our free Wireless Optogenetics eBook, “The Wireless Way: Optogenetics” We explore the basics of in-vivo optogenetics and provide examples. Included are optogenetic suppression of medial prefrontal cortex -> paraventricular nucleus projections and optogenetic activation of parvalbumin inhibitory interneurons in the dorsal-medial prefrontal cortex.

References

Amitrano, A.M. (2021). Optical Control of CD8+ T Cell Metabolism and Effector Functions. Frontiers in Immunology, 12, 666231. https://doi.org/10.3389/fimmu.2021.666231

Catanese, A., Rajkumar, S., Sommer, D., Freisem, D., Wirth, A., Aly, A., Massa-López, D., Olivieri, A., Torelli, F., Ioannidis, V., Lipecka, J., Guerrera, I. C., Zytnicki, D., Ludolph, A., Kabashi, E., Mulaw, M. A., Roselli, F., & Böckers, T. M. (2021). Synaptic disruption and CREB-regulated transcription are restored by K+ channel blockers in ALS. EMBO Molecular Medicine, 13, e13131. https://doi.org/10.15252/emmm.202013131

Duke, Corey G., et al. “Blue Light-Induced Gene Expression Alterations in Cultured Neurons Are the Result of Phototoxic Interactions with Neuronal Culture Media.” Eneuro, vol. 7, no. 1, 2019, https://doi.org/10.1523/eneuro.0386-19.2019. 

Shichida, Y., & Matsuyama, T. (2009). Evolution of opsins and phototransduction. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 364(1531), 2881–2895. https://doi.org/10.1098/rstb.2009.0051

Stierschneider A, Grünstäudl P, Colleselli K, Atzler J, Klein CT, Hundsberger H, Wiesner C. Light-Inducible Spatio-Temporal Control of TLR4 and NF-κB-Gluc Reporter in Human Pancreatic Cell Line. International Journal of Molecular Sciences. 2021; 22(17):9232. https://doi.org/10.3390/ijms22179232

Marek, V., Potey, A., Réaux-Le-Goazigo, A., Reboussin, E., Charbonnier, A., Villette, T., Baudouin, C., Rostène, W., Denoyer, A., & Mélik Parsadaniantz, S. (2019). Blue light exposure in vitro causes toxicity to trigeminal neurons and glia through increased superoxide and hydrogen peroxide generation. Free radical biology & medicine, 131, 27–39. https://doi.org/10.1016/j.freeradbiomed.2018.11.029

What is Microdialysis

What is Microdialysis

Introduction

Microdialysis is a powerful sample collection and administration technique used in scientific research to monitor and analyze the chemical composition of extracellular fluid in various tissues. Microdialysis offers valuable insights into the dynamic changes of neurotransmitters, metabolites, and other molecules within living organisms. In this blog post, we will delve into the intricacies of microdialysis and explore its applications across different fields.

Section 1: Understanding Microdialysis

Principles of Microdialysis

Microdialysis involves the insertion of a semi-permeable probe, known as a microdialysis probe, into the tissue of interest. The probe is perfused with a physiological solution that closely mimics the composition of the extracellular fluid. As the perfusion solution flows through the probe, it creates a concentration gradient that facilitates the diffusion of molecules from the surrounding tissue into the probe. The collected dialysate can then be analyzed to measure the levels of specific analytes.

Probe Design and Materials

Microdialysis probes are available in various designs and materials, each with its own characteristics and applications. The choice of probe design, such as shaft length, diameter, and membrane length, depends on the target tissue. The materials used for the probe membranes can be tailored to suit specific research needs, including biocompatible polymers or glass.

Section 2: Applications of Microdialysis

Neurochemical Research

Microdialysis has revolutionized the field of neuroscience by providing a means to study the dynamic changes in neurotransmitter levels within the brain. Researchers can investigate neurotransmitter release, reuptake, and metabolism in various brain regions. Microdialysis is also invaluable in studying the effects of drugs, diseases, and neuropsychiatric disorders on neurochemical signaling.

Explore Eicom Microdialysis Probes!

Our probes have a 220 µm outer diameter. The probe inlet connects to a syringe pump, which continuously perfuses through the probe.  As the perfusate travels through the probe, analytes cross the membrane and travel out the probe outlet into sample vials on a fraction collector or to a sample loop of an analytical system via a narrow tubing. This valuable tool enables the sampling of rather low molecular weight compounds from within the extracellular space of tissues.

Pharmacokinetics and Drug Development

Microdialysis plays a crucial role in pharmacokinetic studies by allowing researchers to monitor drug concentrations. By continuously sampling from specific tissues, such as brain or muscle, microdialysis provides valuable information on drug distribution, metabolism, and elimination. This data aids in the optimization of drug dosage and formulation during the drug development process.

 

Metabolic Studies

Microdialysis is widely employed in metabolic research to assess tissue-specific metabolism. By measuring the levels of substrate and metabolites, such as glucose, lactate, and amino acids, researchers can gain insights into energy utilization, cellular metabolism, and metabolic disorders. Microdialysis also enables the study of organ-specific metabolism in various tissues, including the liver, adipose tissue, and skeletal muscle.

Environmental Monitoring

Microdialysis finds applications in environmental research for monitoring and analyzing chemical compounds in different environmental matrices. It enables continuous sampling and analysis of pollutants, toxins, or biomarkers in water, soil, air, and biological tissues. Microdialysis contributes to understanding environmental contamination, pollutant fate, and human exposure risks.

Integration with Imaging Techniques

Microdialysis can be combined with imaging techniques, such as fluorescence imaging or mass spectrometry imaging, to provide spatial information about analyte distribution within tissues. This correlation between microdialysis and imaging techniques enables researchers to gain insights into the spatial dynamics and localization of analytes, enhancing their understanding of physiological processes and disease mechanisms. By integrating microdialysis with imaging techniques, researchers can correlate the temporal information obtained through microdialysis with the spatial distribution of analytes. This integration opens up new possibilities for advanced research in fields such as neuroscience, pharmacology, and environmental monitoring.

​​Fluid Intake Precision: The Drinko Advantage

​​Fluid Intake Precision: The Drinko Advantage

Introduction

Precise fluid intake measurement can be extremely important in behavioral neuroscience studies. Accurately measuring the amount of fluid a rodent consumes provides crucial insights into their physiological and behavioral responses. In this blog, we will delve into the significance of precise fluid intake measurement, highlighting the Drinko Measurer. With its enhanced features like precise and accurate drink measurement, leak-free sipper tube, and multiple sizes designed to fit, the Drinko Measurer has improved the way researchers track and analyze fluid consumption in rodent studies.

The Importance of Accurate Fluid Intake Measurement

Accurate measurement of fluid intake is vital in behavioral neuroscience studies for several reasons. Firstly, it allows researchers to evaluate the impact of specific treatments, drugs, or interventions on an animal’s drinking behavior. By precisely quantifying fluid intake, researchers can analyze the effects of these factors on hydration levels, metabolism, and overall physiological state. Secondly, precise measurement enables the detection of subtle changes in drinking behavior that may be indicative of altered cognitive or emotional states. This information is crucial for understanding underlying behavior and for drawing accurate conclusions in neuroscience research.

The Drinko Measurer: Leak-Free Fluid Intake Measurement

The Drinko Measurer has emerged as an ideal tool in the field of behavioral neuroscience for accurately measuring rodent fluid intake. This innovative device provides numerous benefits that enhance the accuracy of fluid intake measurement. Equipped with a double-ball bearing, the Drinko Measurer offers precise volume measurement, ensuring reliable data collection every time. Researchers can track and record the exact amount of fluid consumed by rodents, enabling precise analysis and comparison between experimental groups.

Explore the Drinko Measurer for Precise Fluid Intake Measurements!

The Advantages of the Leak-Free Sipper Tube

The Drinko Measurer incorporates a leak-free sipper tube system, addressing a common challenge in fluid intake measurement. With conventional setups, fluid leakage can lead to inaccuracies and potential confounding factors. However, the Drinko Measurer’s leak-free sipper tube eliminates this issue, ensuring that the measured fluid intake truly represents the consumed amount. This feature enhances the reliability of the data and allows researchers to draw more accurate conclusions regarding the effects of various treatments or experimental conditions on fluid intake.

Versatility and Adaptability with Multiple Sizes

The Drinko Measurer offers researchers the flexibility to adapt to different experimental requirements by providing multiple sizes of sipper tubes. Whether researchers are working with mice, rats, or other rodent models, they can select the appropriate sipper tube size to ensure accurate measurement. This adaptability makes the Drinko Measurer a valuable tool for various behavioral neuroscience studies.

 

Conclusions

Accurate measurement of fluid intake is essential in behavioral neuroscience studies, as it provides valuable insights into the physiological and behavioral responses of rodents. The Drinko Measurer, with its accurate drink measurement, leak-free sipper tube, and multiple sizes, has revolutionized fluid intake measurement. By utilizing this advanced device, researchers can ensure precise data collection, eliminate confounding factors, and enhance the reliability of their findings in behavioral neuroscience research.

Illuminating Neuroscience: The Power of Wireless Optogenetics

Illuminating Neuroscience: The Power of Wireless Optogenetics

Introduction

In the realm of neuroscience research, a groundbreaking technique has emerged known as wireless optogenetics. Wireless optogenetics is a fusion of wireless technology and light stimulation that allows for precise control and manipulation of neural activity in real time. In this blog, we will explore the remarkable applications of wireless optogenetics and its potential to advance our understanding of the brain. We will also delve into the limitations of traditional tethered systems and the numerous benefits offered by wireless optogenetic technology.

The Power of Optogenetics

Optogenetics utilizes light-sensitive opsins to precisely control and modulate specific neurons. By introducing genetically engineered opsins, researchers can manipulate neural activity with remarkable precision, enabling the study of neural circuits, brain activity, and neurological disorders.

Advancing Neurological Research

Wireless optogenetics holds promise in studying neurological disorders like Parkinson’s disease and epilepsy. With wireless technology, precise optical stimulation and neural modulation can be achieved, offering potential therapeutic approaches and novel treatment strategies.

Ready to Illuminate Your Neuroscience Journey? Download Our FREE eBook Now!

Understanding Brain Activity

Wireless optogenetics enables researchers to investigate neural circuits with exceptional accuracy, shedding light on the functional roles and connections between brain regions. This detailed understanding enhances our knowledge of brain function, information processing, and behavior generation.

Wireless Technology and Neurophysiology

Integration of wireless technology in optogenetics revolutionizes neurophysiology. Researchers can study neural activity and behavior in freely moving animals, acquiring real-time data for comprehensive analysis. This integration opens new avenues for studying the brain in natural contexts.

Future Directions

While wireless optogenetics has revolutionized neuroscience, ongoing efforts focus on miniaturizing wireless devices, optimizing power consumption, and developing advanced data analysis techniques. Integration with other cutting-edge technologies holds promise for unraveling the complexities of the brain.

In conclusion, wireless optogenetics with its precise control of neural activity through light stimulation transforms neuroscience research. It provides unprecedented insights into neural circuits, brain activity, and neurological disorders. By combining wireless technology and optogenetics, we are poised to unlock groundbreaking discoveries that will reshape our understanding of the brain.

SFN 2022 Giveaway Winners

SFN 2022 Giveaway Winners

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Questions?

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.

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.

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

Results
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
mice.

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.

Resources

(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
References:
(2) Williams, B., Speed, A., Haider, B: A novel device for real-time measurement and
manipulation of licking behavior in head-fixed mice (2018)
(3) Clark, Nicholas McKinley.: FAST, ACCURATE, AND LOW-COST SENSING OF REWARD
CONSUMPTION AND LICK RESPONSES IN RODENTS (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)