Forgetfulness at the touch of a button: Teleopto Wireless Optogenetics in Science Magazine and The New York Times.

Forgetfulness at the touch of a button: Teleopto Wireless Optogenetics in Science Magazine and The New York Times.

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.

 When interviewed by The New York Times, Prof. Yamanaka explained

“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:

https://science.sciencemag.org/content/365/6459/1308

OptoDroplets: Organelle formation controlled by Teleopto LED arrays

OptoDroplets: Organelle formation controlled by Teleopto LED arrays

What are biomolecular condensates?

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.

You can learn more about optoDroplets at the Brangwynne website.

Teleopto LED arrays are also being used in the development of new optogenetic switches, cardiovascular and nervous system developmental biology, ophthalmology, and photobiochemistry. 

(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.

https://doi.org/10.1038/ncomms5925

(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.

https://www.pnas.org/content/116/35/17330 

(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.

https://doi.org/10.1126/science.1172046

 

New Products at Neuroscience 2019

New Products at Neuroscience 2019

Amuza is showing two new products at Neuroscience 2019

TeleFipho wireless fiber photometry: measure calcium and neurotransmitters in real time in freely moving animals.

Free Maze: The O’Hara Free Maze is a reconfigurable maze that enables researchers to build their own mazes to their own specifications.

We will have a free gift waiting for you at Booth #1502 when you arrive!

Wireless Fiber Photometry: Measuring Neurochemicals In Vivo in Real Time

Wireless Fiber Photometry: Measuring Neurochemicals In Vivo in Real Time

Amuza and Teleopto launch the first commercial wireless fiber photometry system at Neuroscience 2019

Our wireless optogenetic users have frequently asked us if we could provide wireless photometry – we are happy to announce that now we can!

TeleFipho wireless headstages allow your freely behaving animals to move with true freedom, enabling novel experimental approaches with fiber photometry. The 3-gram headstages are optimized for GCaMP and other GFP-based indicators.

TeleFipho includes all of the components required for fiber photometry – light source, filter cube, photodetector, and wireless transmission hardware – in a 3-gram headstage.

What is Fiber Photometry?

Fiber Photometry is a powerful technique for measuring rapid changes in neuromodulators in vivo via fluorescence. It is most commonly used to measure fast (sub-second) changes in concentrations of calcium in freely behaving animals, but it is now also capable of being used to monitor neurotransmitters and other molecules. To use fiber photometry, genetically encoded fluorescent indicators are first expressed at the location of interest. When excited by light of the right wavelength, these proteins fluoresce – but only while they are bound to their target analyte. As local concentrations of the analyte rise and fall, the fluorescence intensity rises and falls in response. Genetically encoded calcium indicators (GECIs), such as GcAMP, have been the mainstay of fiber photometry and also for calcium imaging, a closely related technique. Recently, dopamine indicators (Dlight1, GRABDA) and norepinephrine indicators (GRABNE) have been introduced, and more neurochemical sensors are in development.

To capture this signal in vivo, an optical fiber is implanted in the target region in the animal. The other end of the fiber is attached to the photometry hardware. First, an LED or laser light source passes light through the fiber to excite the indicator proteins in the target region. The resulting fluorescent light then travels back through the fiber to a photodetector, creating a record of the changing concentrations of the analyte. Careful filtering and splitting of the light traveling through the fiber optic are required to separate the light used for excitation from the fluorescence being sent to the photodetector.

Why use Fiber Photometry?

The most frequent use is to measure changes in calcium levels at synapses as a proxy for changes in neural activity, helping researchers discern the links between behavior states and the firing patterns of neurons. But the same technique is also used to monitor the activity of GPCRs and ion channel drug targets.

When used with freely moving animals, fiber optic tethers can be problematic. The cable can prevent animals from using exercise wheels or shelters or getting tangled in complicated environments, limiting behavioral testing. Cables can also cause artifacts when used with video tracking software. For example, the cable often continues to sway after the animal has stopped moving, making it difficult to recognize freezing behavior during fear conditioning studies. Placing all of the necessary components for fiber photometry in a small lightweight headstage ends these problems.

TeleFipho has been tested with both mice and rats. The data above shows stress-induced (tail pinch) changes in GCaMP signals from hypothalamic orexin neurons in mice. GCaMP is a genetically encoded calcium indicator often used to monitor calcium dynamics. Data is Courtesy of Dr. Daisuke Ono in the Akihiro Yamanaka Lab, Nagoya University.

TeleFipho has been tested with both mice and rats. The data above shows stress-induced (tail pinch) changes in GCaMP signals from hypothalamic orexin neurons in mice. GCaMP is a genetically encoded calcium indicator often used to monitor calcium dynamics. Data is Courtesy of Dr. Daisuke Ono in the Akihiro Yamanaka Lab, Nagoya University.[/caption]

Shrinking the components for fiber photometry has a bonus: it also allows us to shrink the price. Telefipho starts at roughly half of the cost of other commercial fiber photometry systems.

Please stop by our booth during SfN 2019 to ask for a demonstration and visit our product page for more information.

Questions?

How to Improve Microdialysis Throughput and Success Rates

How to Improve Microdialysis Throughput and Success Rates

There are many ways to speed up your microdialysis experiments and ensure their success.

During surgery:

  • Stereotaxic frames with digital readouts are much quicker to use than ones still equipped with vernier scales: the instant readout and ability to zero each axis speed up each movement of the manipulator. Digital readouts are available as a retrofit for many older stereotaxic frames. Additionally, dual manipulator stereotaxic frames can speed up each surgery by keeping multiple tools accessible at all times.
  • After the initial incision, scratch the surface of the skull with a scalpel: cement will bond more tightly to the roughened surface.
  • Wipe the skull with hydrogen peroxide to help dry it and then wipe with epinephrine to prevent bleeding. For implantation to be successful, the surface of the skull must be dry. If the surface is still damp when cement is applied, the trapped moisture can lead to infection and necrosis of the skull.
  • Switching to UV cure cement lets you avoid mixing a new batch (powder plus solvent) of acrylic cement each time your previous batch hardens. A simple UV light hardens the cement when you need it to – not before.

For sample collection:

  • Use 96 well plates to collect samples instead of individual sample tubes. This eliminates capping, labeling, and handling of individual tubes as samples are collected and moved to storage. Used in conjunction with well plates, self-closing plate seals such as silicone sealing mats and cap mats will protect samples from evaporation and contamination.
  • Use the best fraction collector. The Amuza FC-90 collects from up to four animals/probes simultaneously and never skips a sample. Samples can be collected into 96 well plates or racks of individual tubes, and are kept refrigerated throughout.

And finally – Ask Amuza!
Amuza (formerly Eicom USA) has decades of experience in microdialysis and HPLC-ECD, and we are always ready to help.

 

Eicom AtmosLM Microdialysis Used in Developing Pharmacokinetic Models of Therapeutic Antibody Distribution in the Brain

Eicom AtmosLM Microdialysis Used in Developing Pharmacokinetic Models of Therapeutic Antibody Distribution in the Brain

The brain is a challenging target for therapeutic monoclonal antibodies (mAbs), nanobodies, antibody drug conjugates (ADCs), and other drugs. The blood brain barrier prevents many drugs with otherwise good absorption profiles from crossing into the brain, and also complicates attempts to model how drugs are distributed within the brain.

Prof. Dhaval Shah and PhD student Hsueh-Yuan Chang of the University at Buffalo study the pharmacokinetics/pharmacodynamics (PK/PD) of therapeutic antibodies and ADCs. Their lab recently used the Eicom AtmosLM (large molecule) microdialysis system as a way to quantitate a mAb in multiple brain regions simultaneously, generating data to underpin pharmacokinetic models for the disposition of mAbs in rats (1,2). They found that tissue homogenate and lumbar cerebrospinal fluid samples do not make good proxies for predicting mAb concentrations at their sites of action within the parenchyma of the brain. They also found that the lateral ventricles and the blood-CSF barrier may be an important route for mAb entry.

How Large Molecule microdialysis works

AtmosLM is a push-pull microdialysis system for measuring the levels of large proteins and peptides, as opposed to the catecholamines and other small molecules typically measured by microdialysis. AtmosLM features unique probes that incorporate vents to equalize the pressure inside the membrane of the probe with the outside atmosphere. This prevents ultrafiltration and yields more consistent analyte recovery rates than other push-pull systems. It has been widely used to study levels of Abeta, Tau, synuclein, lipidated ApoE particles, cytokines, and other molecules. Recently, the Derendorf lab
(U of Florida) used AtmosLM to determine tissue interstitial concentrations of mAbs in liver, skin, kidney, and muscle after IV dosing to aid in their development as anticancer drugs.

Microdialysis based PK modeling

Amuza spoke with Hsueh-Yuan (Luke) Chang about how he used AtmosLM in this project, and he also shared several tips for other users of AtmosLM.

Amuza: Could you explain how your PBPK (physiologically-based pharmacokinetic) model can be used by those studying the use of mAb based drugs in the brain?

Hsueh-Yuan: Our current version of the PBKP model was developed to capture nonspecific mAb distribution in the brain and different regions of the brain. Additionally, it can help to quantify the correlation between mAb concentrations in brain CSF and ISF. It may also help to quantify the amount of mAb entering brain parenchyma versus brain CSF compartments.
While the nonspecific mAb PBPK model has not incorporated target binding or receptor-mediated transcytosis yet, both novel delivery mechanisms and target binding kinetics can be mechanistically added into the current basic version of the PBPK model.
The final version of the PBPK model for mAbs may provide an a priori prediction of mAb distribution in the human brain once kon/koff of values of specific mAbs and receptor/target concentrations in the brain have been included. This prediction could be tested in rodents and primates.

Amuza: Central nervous system (CNS) concentrations of mAbs are often determined by taking whole brain homogenate samples or cerebrospinal fluid (CSF) samples from the lumbar region. What are these methods missing?

Hsueh-Yuan: They do not provide direct information of the mAb concentration at the site-of-action as we mentioned in the introduction of the paper. There are many studies suggesting mAb concentrations are different between CSF and ISF.
More importantly, some studies have reported mAb accumulation within brain capillary cells, which may utilize endogenous receptor binding to enhance brain uptake of mAbs.

Shah lab insights for using AtmosLM

Amuza: I’d also like to ask you a few questions about using our AtmosLM system.

You used siliconized sample tubes to prevent adsorption of antibodies in your samples to the plastic. Would blocking the tubes by rinsing with BSA work as well?

Hsueh-Yuan: Yes. However, the storage of low concentration IgG microdialysates requires 0.1-0.15% BSA. BSA is compatible with ELISA. For LC/MS, the BSA method should be replaced.

Amuza: Do you think endogenous IgG could be used similarly to an internal standard to suggest whether or not a microdialysis experiment is working correctly?

Hsueh-Yuan:  Yes, ELISA methods can quantify rat, mouse, or human IgG specifically. They can serve as an endogenous IgG reference, a proxy for calculating in vivo recovery of the mAbs themselves. Hemoglobin can be used, too.

Amuza: During in vitro tests, you were very careful when balancing the flow between the syringe pump (push) and the peristaltic pump (pull), adjusting the peristaltic pump until the ratio of fluid pumped in/out of the probe stayed in the range of 97 – 103%.

What happens if fluid recovery is outside of this range?

Hsueh-Yuan: Then convection [bulk flow of solutes and solvent across a membrane due to a pressure imbalance] will happen.

Amuza: This is indeed a problem. Your paper (1) found that recovery rates were strongly changed when the flow was not properly balanced. This data is available in the supplementary material.

How did you measure the amount of fluid recovered in each sample?

Hsueh-Yuan: By measuring their net weight.

Amuza: With AtmosLM probes, this can also be accomplished by visually monitoring the flow exiting the probe during in vivo experiments. If the peristaltic pump is pulling fluid out of the probe faster than the syringe pump is pushing fluid in, air will enter the system through the vent in the probe and be visible as bubbles in the tubing. If instead the syringe pump is pushing more fluid than the peristaltic pump is removing, the excess fluid will exit the probe through the vent hole. The vent hole is downstream from the membrane, and does not interfere with microdialysis.

What suggestions do you have for others using AtmosLM to study antibody concentrations?

Hsueh-Yuan: 

  • Endogenous IgG or another internal reference should be measured to validate that the BBB is intact.
  • Fresh CSF perfusion buffer should be used. BSA may precipitate if the CSF is left sitting at room temperature.
  • Due to the instability of low concentrations of antibodies in microdialysate, samples should be analyzed ASAP. Alternatively, standards should be diluted to their final concentrations and stored together with the samples until analysis.
  • Always check the inlet and outlet of the probe before connecting the probe to the push-pull system.

Amuza: Do you have future projects in mind for large molecule microdialysis?

Hsueh-Yuan: Yes. We have been working on several projects using AtmosLM microdialysis.

 

  1. Chang, H. Y., Morrow, K., Bonacquisti, E., Zhang, W., & Shah, D. K. (2018, August). Antibody pharmacokinetics in rat brain determined using microdialysis. In MAbs (Vol. 10, No. 6, pp. 843-853). Taylor & Francis.https://doi.org/10.1080/19420862.2018.1473910
  2. Chang, H. Y., Wu, S., Meno-Tetang, G., & Shah, D. K. (2019). A translational platform PBPK model for antibody disposition in the brain. Journal of pharmacokinetics and pharmacodynamics, 1-20.https://doi.org/10.1007/s10928-019-09641-8
  3. Jadhav, S. B., Khaowroongrueng, V., Fueth, M., Otteneder, M. B., Richter, W., & Derendorf, H. (2017). Tissue distribution of a therapeutic monoclonal antibody determined by large pore microdialysis. Journal of pharmaceutical sciences106(9), 2853-2859. https://doi.org/10.1016/j.xphs.2017.03.033

The Amuza FC-90 is a game changer for any microdialysis experiment:

4 channel operation: collect from up to 4 animals simultaneously.

Ideal for large and small molecule microdialysis.

Refrigerated storage in 96 well plates.

Small footprint: 7″ wide.

Acetylcholine Neurochemical Involvement in Gulf War Illness

Acetylcholine Neurochemical Involvement in Gulf War Illness

For approximately 200,000 US veterans, the 1991 Persian Gulf War marked the beginning of their experience with Gulf War Illness (GWI). GWI encompasses a cluster of chronic symptoms including memory and cognitive problems, fatigue, and fibromyalgia.

GWI has long been associated with a combination of several possible contributory factors: the stress of deployment, altered immune function, and exposure to acetylcholinesterase inhibitors (AChEI), but the exact cause or causes have remained elusive. The AChEI pyridostigmine bromide (PB) was administered to soldiers as a prophylactic against the risk of nerve agent weapons, but many veterans were also exposed to AChEI based pesticides, further complicating the etiology of this illness.

To elucidate the relationship between these factors, Dr. Victoria Macht, her advisor Prof. Lawrence Reagan, and colleagues at the University of South Carolina School of Medicine studied rats exposed to pyridostigmine bromide and repeated restraint stress. The rats were then given either an immune challenge or an acute immobilization stress challenge during in vivo microdialysis. It is the first study to use an in vivo method (microdialysis) to show that PB changes the response of the central cholinergic system to both stress and immune challenges, and does so in a brain region specific manner.

By measuring acetylcholine levels via microdialysis and subsequent HPLC-ECD, they found that cholinergic responses were attenuated in the PFC and hippocampus after immobilization stress. Lipopolysaccharide (LPS) was administered as an immune challenge, after which cholinergic responses were attenuated in the hippocampus but not the PFC. These results indicate that PB and stress interact to shift the cholinergic response to future psychological and immunological stressors, providing a potential mechanism for the persistent and exacerbated cognitive symptoms evidenced in soldiers with GWI.

__

Mike Churchill: What story do the different responses to the immune challenge and the immobilization challenge tell?

Victoria Macht: By using two different types of challenges, we were able to test both the diversity and consistency of effects of PB and stress on the cholinergic system. LPS is a novel challenge which specifically elicits a response from the innate immune system. The immobilization challenge is more of a psychological stressor, and as it shares some similar qualities with the prior restraint stress, this allowed us to test if rats with PB and restraint stress had impaired neurochemical adaptations to recurrent stressors.

MC: How might these results relate to changes in fear memory and cognitive function?

VM: ACh is an important regulator for a variety of factors in fear memory including coordination of local circuits to help with sensory and cortical processing of stimuli as well as the consolidation process. Interestingly, regional differences in the cholinergic response of the PFC and hippocampus to immobilization stress suggested that PB impairs cortical processing of novel stressful stimuli and impairs the neurochemical adaptation to recurrent stressful stimuli. In our fear conditioning studies, we similarly found impairments in the way PB and stress interacted to impair context and cue related retrieval. This suggested to us that impairments in the function of the cholinergic systems impacts a variety of psychological stressful stimuli, indicating that this is a global deficit in cognitive function rather than a specific deficit to only one type of stressor.

MC: How do the microdialysis results relate to the tests for inflammation you ran?

VM: ACh is really fascinating because while it is not only central in learning and memory, it is also an important negative regulator for the inflammatory response via α7 nicotinic ACh receptors. We found that PB blunted the central cholinergic response to an innate immune challenge, which could suggest an exacerbated chronic inflammatory response in the brain. Interestingly, these microdialysis results for acetylcholine parallel some of our findings with peripheral inflammatory markers. Peripheral levels of c-reactive protein were elevated after the LPS challenge in rats which had received PB, suggesting a dysregulated inflammatory response. While we need to confirm these results with cytokine levels in the brain, our results suggest that impaired cholinergic feedback to inflammatory stimuli could underlie some of the changes in the sensitivity of the immune system which are evident in clinical populations with GWI.

MC: Does PB have to cross the BBB to cause these effects?

VM: It does not. There has been a big debate on this topic. One suggestion was that stress caused a leaky barrier, allowing PB to get through. However, tests on this have been inconsistent on this. What our studies demonstrate is that PB changes the function of the central cholinergic system regardless of whether it is able to get through the BBB.

MC: What will be the next steps for this project?

VM: Prof. Reagan will continue the project: measuring cytokine responses in the brain to see if they match peripheral cytokine responses. There is also an opportunity to see if aging exacerbates the decline of the cholinergic responses and cognitive deficits in our model of GWI. The goal would be to see if animal models of GWI can predict further changes in veterans as they age, and plan treatment accordingly. We have a unique opportunity with this population for the preclinical research on treatments to get ahead of the patient population as they age.

MC: How did you like using the Eicom HTEC HPLC-ECDs in Prof. Jim Fadel’s lab?

VM: It is amazing! I can’t imagine having done these projects without it, and I miss using it.  We used the system daily for two years to measure acetylcholine without any real problems. It made my dissertation a much more pleasant experience!

MC: Had you used HPLCs before using the HTEC?

VM: We used a different system before but it was not reliable, so when it was working people felt they had to immediately run all of their samples before it went down again, and watch it all of the time when it was running.

MC: How many samples do you think you ran over the course of this project?

VM: That makes my head spin! We looked at both ACh and glutamate, in two brain regions, each rat underwent microdialysis 2 separate days, there were approximately 8 animals per group, and 4 groups. So at least 3500 samples – plus the pilot study! Plus there were other studies going on during this time which were also using the HTEC.

MC: Where is your career taking you next?

VM: I am now doing a postdoc at UNC Chapel Hill, working with Prof. Fulton Crews, studying the long term effects of binge drinking in adolescents. Interestingly, while this is a different clinical population, changes in the cholinergic system and innate immune system are also common features here.

__

The article appears in the April issue of Brain, Behavior, and Immunity:

Pyridostigmine bromide and stress interact to impact immune function, cholinergic neurochemistry and behavior in a rat model of Gulf War Illness
V.A. Macht, J.L. Woodruff, E.S. Maissy, C.A. Grillo, M.A. Wilson, J.R. Fadel, L.P. Reagan
doi: 10.1016/j.bbi.2019.04.015

Optogenetics Resources, Guides, and Protocols

Optogenetics Resources, Guides, and Protocols

 

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.

The Deisseroth lab (d-lab) website also hosts

  • 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 paper serves 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.

These media and supplements are now available from Cell Guidance.

Non-neuronal Optogenetics

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:

Impaired clearance of GABA in the amygdala may underpin many cases of alcohol addiction.

Impaired clearance of GABA in the amygdala may underpin many cases of alcohol addiction.

After being conditioned to drink alcohol, 15% of rats continue to seek alcohol after being given the choice of switching to sweetened water. This group of rats shows a high motivation to pursue alcohol even when it is paired with a negative stimulus such as an electric shock, mimicking the compulsion as well as the frequency of alcohol-related problems in humans. Further examination of this group revealed the GAT-3 γ-aminobutyric acid transporter protein was significantly downregulated in the amygdalas of these rats, leading to decreased clearance of γ-aminobutyric acid (GABA) from synapses in this region.

To confirm this model, the Heilig group of the Linköping University in Sweden then downregulated GAT-3 in the amygdala in rats which until that point showed no preference for alcohol. As the injected vector began to downregulate GAT-3, the rats showed a stronger and stronger preference for alcohol over sweetened water.

This model also seems to hold true for humans: postmortem examination of human subjects showed lower GAT-3 expression in the amygdalas of those with alcohol dependence.

Monitoring GABA levels in research animals are what we do: please visit our Neuroscience products page to learn more about microdialysis and HPLC-ECD analysis for GABA and other analytes.

The study is being lauded not only for proceeding from basic research to translational significance in humans but also for its addition of choice to the basic experimental design. Allowing the research animals to choose between self-administering alcohol or switching to another reward after the conditioning period required a more lengthy and larger scale project (over 600 rats), but it revealed how the animals should be grouped together for further analysis.

While this study indicates a causal relationship between a GABA transporter and alcohol dependence, it may not point directly to the best drug target for addiction therapy. Increasing GABA transport back into neurons in the amygdala may prove more difficult than other ways of controlling GABA levels in the region.

The Heilig group of the Linköping University in Sweden presented the results at the Research Society on Alcoholism meeting in San Diego and subsequently in the journal Science:

A molecular mechanism for choosing alcohol over an alternative reward
Science  22 Jun 2018: Vol. 360, Issue 6395, pp. 1321-1326, DOI: 10.1126/science.aao1157

Questions?

Measurement of Nitrate and Nitrite in Biopsy-Sized Muscle Samples

Measurement of Nitrate and Nitrite in Biopsy-Sized Muscle Samples

Researchers at Indiana University Purdue University Indianapolis recently presented an improved method using the ENO-30 to determine nitrite and nitrate levels in human skeletal muscle at the American College of Sports Medicine annual meeting. Muscle tissue has been determined to be an important reservoir of nitrite and nitrate and thus may serve as an endogenous source for nitric oxide production in the body. However, analysis of human muscle biopsy samples for NOx has until now been limited by their small size. A recent study1 reported nitrite was below the limit of detection by chemiluminescent analysis even when assaying up to 40 mg of tissue.

Assoc. Prof. Andrew Coggan’s lab validated a method for reproducibly determining nitrite and nitrate in samples as small as 5 mg using the ENO-30. The levels of NOx so determined were consistent with literature results from much larger muscle tissue samples analyzed by other methods.

Results of the study include:

  • The ENO-30 HPLC proved to be ideally suited for the present application, with excellent baseline stability (Fig. 1), sensitivity (limit of detection = 0.06 ± 0.01 pmol; limit of quantification = 0.20 ± 0.03 pmol; n=6 standard curves) (Fig. 2), and linearity of response (Fig. 3). A representative sample chromatogram is shown in Fig. 4

Figure 2. Sensitivity of HPLC (0.3 pmol NO2- standard).

  • Pulverization of tissue at liquid N2 temperature followed by extraction in 50 µL methanol + 0.5% Triton X-100 resulted in the highest NO3- and lowest NO2- contents and the least variability. (Fig. 7)

Figure 7. Reproducibility of extraction methods.

  • NO2- content of tissue extracts stored at -80°C increased over time, suggesting residual xanthine oxidoreductase (XOR) activity (Fig. 8). Inclusion of 0.1 mmol/L oxypurinol in the extraction medium completed blocked this increase.

Researchers at Indiana University Purdue University Indianapolis recently presented an improved method.

They further determined that the method may apply for nitrate analysis in samples as small as 5 ng.

The Coggan lab’s method should become useful in studies of NOx metabolism in muscle tissue in response to exercise and dietary interventions – and make participation in these studies much less painful!

The poster can be viewed here.

 

1Nyakayiru et al. J Appl Physiol 2017; 123:637-644

Questions?

Eicom at SfN 2016

Eicom at SfN 2016

Please join the Eicom USA team at SfN 2016 in our very own hometown of San Diego.

Stop by our booth #1532 to learn more about our products or for a simple hello.

We look forward to seeing you:

Thank you for attending FENS 2016

Thank you for attending FENS 2016

We had a booth at Federation of European Neuroscience held in Copenhagen, Denmark.
For this time, Mike Churchill joined the show from our US Office.

We had great discussions with our customers in Europe. Thank you to all who stopped by our booth!