Melanin-concentrating hormone (MCH) neurons are unlike most neurons: they are most active during sleep. Scientists have studied their role in regulating sleep and feeding behavior for some time, but the Yamanaka lab at Nagoya University in Japan has found that they may also have a role in preventing the consolidation of memories during sleep.
Prof. Yamanaka (a co-developer of Teleopto) found that MCH neurons can suppress neurons in the hippocampus responsible for memory consolidation. His lab confirmed this role by using
Teleopto Wireless Optogenetics: blue light and channelrhodopsin 2 were used to activate MCH neurons; green light/archaerhodopsin were used to inhibit them. This was done bilaterally during both memory consolidation (REM sleep) and awake periods. Teleopto was used so that the animals were able to move freely and interact naturally with objects during Novel Object Recognition (NOR) tests.
When mice had MCH neurons activated during sleep, their ability to remember events decreased: they forgot which objects they had encountered before sleeping and treated them the same way they treated novel objects. Conversely, when their MCH neurons were inhibited they were able to remember which objects they had already interacted with. They ignored the familiar objects and explored the novel objects instead.
When Teleopto was used to illuminate MCH neurons during awake periods, there was no effect on hippocampal-dependent memory.
“These results suggest that hypothalamic M.C.H. neurons help the brain actively forget new information that is not important.” And because the neurons are most active during R.E.M. sleep, they may explain why humans usually do not remember their dreams when they wake up. “The neurons may be clearing up memory resources for the next day,” Dr. Yamanaka said.
The article, “REM sleep–active MCH neurons are involved in forgetting hippocampus-dependent memories.” is available at:
Biomolecular condensates are a unique class of organelles: they have no membranes. They can form, merge, split, and disappear in minutes, temporarily creating local incubators and assembly lines with properties very different from the bulk of the cells surrounding them. The local high concentrations of proteins and polynucleotides inside these condensates can both speed up and interfere with reactions, challenging the researchers trying to understand the rules of cell biology.
Some condensates, such as the nucleolus and Cajal bodies, were first observed over a century ago, but others such as processing bodies, PML bodies, and paraspeckles were only discovered recently. It is only within the past few years that researchers have begun to understand that these organelles all share a common organizing principle: protein association drives the formation of gels which coalesce into the organelles themselves, which then behave according to the classic rules of phase separation and phase transition. These organelles condense in much the same way water vapor condenses into droplets on a window.
Why study biomolecular condensates?
This new understanding has led to condensates becoming a target for drug design. Dewpoint Therapeutics launched earlier this year, based on studies of stress granules. They seek to prevent temporary condensates of FUS protein from congealing into permanent aggregates, a driving force in amyotrophic lateral sclerosis (ALS).
Liquid-liquid phase separation also has a role in gene expression: transcription factors have been found to rely on segregation inside condensates to initiate and control RNA production, yielding new targets for cancer therapies. The kinetics of ribosomal RNA processing is also proving to be dependent on the extent of gelation of the nucleolus.
Teleopto LED arrays and Biomolecular condensates
Clifford Brangwynne, Macarthur Fellow and Assoc. Prof. at Princeton University uses light to control the formation of condensates. Once activated by light, proteins like Cry2olig1 oligomerize within seconds. By fusing Cry2olig to an RNA binding protein that drives condensation in the nucleus (NPM1), the BW lab created optoDroplets: light activated condensates held together by a meshwork of protein and nucleic acids.
Blue light from a Teleopto LEDA array causes these CRY2 fusions (opto-NPM1) to coalesce into a meshwork of proteins capable of turning the nucleolus of a cell into a tightly linked gel2. The lab tunes the properties of the optoDroplets by adjusting the brightness: more light leads to more self-association and smaller pores in the meshwork. As the pores shrink, small proteins can still move through the hydrogel but larger molecules and complexes become trapped. This model allows the Brangwynne lab to study the effect of viscoelasticity on the formation of ribosomes and the processing of rRNA with just the press of a button. In a recent PNAS paper, they found that increasing the gelation of the nucleolus leads to the accumulation of larger rRNA precursors, while smaller precursors are depleted.
After the light is turned off, the condensates typically degenerate within 5 minutes. Fixing the cells while they are still illuminated allows the optoDroplets to be imaged and studied later, as shown in the figure below:
Incubator-compatible Teleopto LED arrays are tools designed for doing in-vitro optogenetics on 96 well plates. The arrays are available in wavelengths from UV to infrared and can be controlled by most pulse generators.
Postdoc Jorine Eeftens said that the Brangwynne lab used to use microscope mounted lasers to make condensates, but that only let them focus on a few cells at a time. The LEDA array allows them to activate many cells at once, greatly improving throughput in the lab. “We use it routinely, every day. We love working with it, the [LEDA] array allows us to use lots of cells, and then fix them for study. It’s our high throughput system.”
Biomolecular Condensates and Teleopto at the Woods Hole Physiology Course
The Woods Hole Marine Biology Laboratory discovery courses are intense, full-immersion summer courses for graduate students and postdocs. Students brainstorm, design and carry out their own projects – which frequently lead to publications. Ten years ago during a course led by Anthony Hyman and Brangwynne, then a postdoc in the Hyman lab, a project showed that P-granules behave like oil droplets when shearing forces are applied. The initial result from the Woods Hole class was followed up by Hyman and Brangwynne at Max Planck Institute, leading to a publication for both the students and the instructors. The paper shows that p-granule behavior follows the classic rules of phase separation and hinted at how this process could be involved in many more aspects of cellular behavior than previously thought3.
Coming full circle, this past summer Prof. Brangwynne and his postdocs led one of the Woods Hole course rotations and focused on the role of condensates in the nucleolus. They brought a LEDA array so that students could form optoDroplets in incubators during the class.
(1) Taslimi, A., Vrana, J. D., Chen, D., Borinskaya, S., Mayer, B. J., Kennedy, M. J., & Tucker, C. L. (2014). An optimized optogenetic clustering tool for probing protein interaction and function. Nature communications, 5, 4925.
(2) Zhu, L., Richardson, T. M., Wacheul, L., Wei, M. T., Feric, M., Whitney, G., … & Brangwynne, C. P. (2019). Controlling the material properties and rRNA processing function of the nucleolus using light. Proceedings of the National Academy of Sciences, 116(35), 17330-17335.
(3)Brangwynne, C. P., Eckmann, C. R., Courson, D. S., Rybarska, A., Hoege, C., Gharakhani, J., … & Hyman, A. A. (2009). Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science, 324(5935), 1729-1732.
We would like to share some of the many resources our customers have found useful for planning their optogenetics projects. Please post comments if you know of more resources we should include – we will update this list regularly.
Starting From Scratch
For neuroscientists, Karl Deisseroth’s Optogenetics Resource Center is perhaps the best first stop when planning your experiment. The Deisseroth lab provides optogenetics related DNA cassettes and vectors to the optogenetic research community, as well as training and workshops for in in-vivo optogenetics on campus at Stanford University.
A light power vs distance attenuation calculator for brain tissue,
Chromophore, DNA sequence, and vector databases,
Information on requesting the many viruses and DNA provided by D-lab to other researchers.
Links to protocols for many optogenetics based methods.
Mapping Neural Circuits
Karl Deisseroth’s 2016 Cell paperserves as both a primer on mapping neural circuits via optogenetics and a review of the many optogenetic switches available for the task.
Finding the Best Switch
If you are hunting for the right optogenetic switch for your project, the OptoBase website provides curated databases of optogenetic techniques, but the true value is in the indexing, tagging, and online tools the BIOSS team created to accelerate your search. For example, In the publication search, filters such as “multichromatic” return only papers which combine multiple optogenetic switches within a single optogenetic system and “Exclude Background” let you exclude basic research on photoreceptors.
The OptoBase’s “Find the Application” tool is a publication selector allowing you to tick off optogenetic uses (e.g. “Control of vesicular transport” AND “control of second messengers”) and returns only those publications which actually used those methods – as opposed to just mentioning them in the discussion section. Publications are tagged and updated weekly. The Optobase is a collaborative project of the BIOSS Centre for Biological Signalling Studies.
Preventing Phototoxicity during in-vitro Experiments
Phototoxicity presents the risk of introducing artifacts or cell death in both in-vitro and in-vivo experiments, particularly when the illumination wavelength is lower than 500 nm. For in-vitro experiments, Káradóttir et. al. found that careful choice of the culture media components can prevent many of the issues from occurring in neuronal cell cultures. In particular, removing riboflavin, thyroxine, and triiodo-1-thyronine and including additional antioxidants increases cell survival considerably.
For non-neuronal optogenetics, EMBL has placed a short introductory course online.
Transgenic Models – Ready to Go
While many companies supply strains of mice and rats ready for transgenic manipulation to introduce optogenetic switches, the Jackson Laboratory provide many strains of transgenic mice already expressing channelrhodopsin (CHR2), archaerhodopsin (Arch), and halorhodopsin (NpHR).
Turnkey In-Vitro and wireless In-Vivo Optogenetics Systems
Amuza provides Teleopto wireless systems for in-vivo optogenetics in mice and rats as well as LED arrays for in-vitro optogenetics in culture plates.
Teleopto wireless comprises a detachable, rechargeable headstage (receiver) and LED fiber optic implants which together weigh as little as 1.3 grams. Our starter kits are turnkey solutions for your first experiment, including receivers, LED implants, remote control, charger, and stereotaxic adapters.
Teleopto LED arrays are normally made for illuminating 96 well plates inside incubators, but can be customized to many different sizes and well configurations.
Both systems are available in colors across the spectrum including UV, violet, blue, yellow, green, red, far red, and IR. Multicolor options are also available. Please visit the Amuza site for more information:
We are excited to announce that Amuza Inc have been awarded an R41 Technology Transfer grant by the National Institute of Neurological Disorders (NINDS/NIH, USA) to develop “a unified system of wireless optogenetics and brain microdialysis”. The grant proposal was based on research which combined Amuza’s HPLC-ECD, microdialysis, and optogenetics instruments.
The funding is for Phase I (prototype development and validation) of a Small Business Technology Transfer (STTR) project in collaboration with Drs. Nobuyoshi Suto (The Scripps Research Institute), Thomas Jhou (Medical University of South Carolina) and Matthew Buczynski (Virginia Tech).
After research and commercialization phases have been completed the final opto-dialysis system will be available from Amuza as well. We are currently undertaking the pilot experiments in freely moving animals (both rats and mice) using our opto-dialysis prototypes. We will post the results as they become available. Please stay tuned!
While still a very new technology, results have been published in Neuron and Molecular Brain showing Teleopto’s utility in experiments that would be difficult to perform with traditional tethered systems:
Dr. Murayama’s group used Teleopto to provide bilateral illumination to the mouse somatosensory cortex during experiments in Y-mazes and place preference cages. This allowed them to suppress tactile sensory circuits (M2 to S1 axons), thus degrading accurate sensory perception of tactile surfaces.