Scarce Microdialysis Goods May Delay Neuroscience Research

Scarce Microdialysis Goods May Delay Neuroscience Research

Even with thorough planning, global supply chain issues have caused delays in nearly every industry, including lab supplies. Microdialysis probes and other consumables, such as pipette tips, prove difficult for researchers to source due to the globally stressed transit structures. Manufacturing and shipping are complex processes. Combined with labor issues and jammed cargo ports, the pandemic is exposing the shortcomings of a weak supply chain.

Eicom, a world leader in microdialysis and electrochemical detection, is headquartered in Japan. In business since 1986, Eicom would probably be seeing the same delays as other microdialysis providers, except for a well-planned overseas inventory structure.

Eicom’s partner in North America, Amuza, houses microdialysis inventory in San Diego, California.

“Most often Amuza is able to ship microdialysis probes with one or two days,” says CEO, Shinji Azuma. “Serving researchers is our priority.”

A broader reach for materials and a network of partners is thus far succeeding in experiencing minimal supply chain issues for Amuza. Customer Service teams can excel through staying alert for order status changes, managing stock, and keeping customers well informed. Amuza also manages the supply chain by making some items at the San Diego office, such as microdialysis tubing with pre-installed connectors. By customizing microdialysis tubing then preassembling connectors on-site, Amuza has been able to mitigate the pandemic’s effects on sourcing, giving researchers a faster turnaround.

“Throughout the pandemic, we have kept up with microdialysis product orders,” says Office Manager, Ai Cole. “If we do have to inform a customer about a delay, it has been for customs. Fortunately, we have not had the delays we hear others are experiencing.”

Business news outlet, Quartz reports, more than 2 months out, “It’s already too late to ship good to the US in time for Christmas.” Customs has been swamped this past year, and the delays are affecting both holiday gifts and lab essentials needed for research projects.

How Bad Are Delays in Microdialysis Probes?

One researcher mentioned they eventually had to switch vendors, after calling 3 different vendors trying to source microdialysis equipment.

Another said they “had to wait 3 months for a microdialysis syringe pump”.

In other conversations, users have mentioned that some models of microdialysis probes or microdialysis accessories could be back-ordered for months.

When research projects aren’t able to source products necessary for research, science suffers, medicine suffers, humanity suffers. It sounds extreme, but each scientific article published is a result of years of research using lab tools necessary to complete the work.

Without the goods, our scientists lack the tools needed to advance knowledge and discovery that advances human lives.

Most microdialysis probes and accessories are still in stock at Amuza. However, when some inventory does reach low levels, Amuza has still been able to get partial orders out the door quickly. For researchers, Cole suggests, “Ask your supplier. Some suppliers may be able to help you with partial shipments to keep your research going.” At least neuroscience research can stay moving and not halt progress.

What lab products are you finding most difficult to source? Let us know in the comments.

Successful fiber photometry in mazes and other complex environments

Successful fiber photometry in mazes and other complex environments

Fiber photometry is most commonly used to monitor activity and chemical signaling at a specific location in the brains of freely moving animals. The technique uses fluorescent sensor proteins to report on the changing local concentration of calcium and neurotransmitters in real-time. To capture the fluorescent signal, an optical fiber is implanted in the brain with the tip just above the region of interest.

Fiber photometry: Wireless vs patch cord

Traditionally, a fiber optic patch cord (patch cable) is used to both deliver the excitation light to the animal and return the fluorescent signal to a fluorescence cube and a detector outside the cage. However, patch cords can create severe limitations for researchers. Sometimes the cord can directly affect the behavior of the animals: mice become confused, tilt their head, gnaw on the cord, or otherwise react to its presence. The patch cord can also run into objects in the environment, limiting the animal’s ability to move or carry out tasks. Patch cords can also introduce noise and artifacts into the signal when used with fiber photometry. Patch cords also complicate scoring of freezing behavior in fear conditioning experiments. Even after an animal freezes (stops moving), the patch cord can keep swaying, and some video tracking software will confuse this with the continued movement of the animal.

Many mazes, for example, elevated zero mazes and elevated plus mazes, have high walls which can trap the cable as a mouse or rat moves from an open area to an enclosed area.

Zero Maze

Elevated Plus Maze

Combining fiber photometry with behavioral measures for anxiety

Rodents typically prefer enclosed locations, and become anxious in unenclosed, wide-open spaces will limit their time spent exploring wide-open spaces. Anxiety levels play a strong role in how willing mice are to explore the unenclosed areas of a maze, so the zero maze is frequently used to study if a drug or other intervention is anxiolytic or anxiogenic. But a patch cord catching on the end of a wall could prevent a mouse from returning to the enclosed alley, so the researcher may have to be present to manipulate the cord.

Patch cords hanging from above a maze can get caught in doorways and at the ends of alleys.

In contrast, users have found mice fitted with wireless headstages such as those used with Amuza fiber photometry, optogenetics, and EEG have no trouble navigating mazes, and users report that the animals behave naturally.

For example, the Dimitrov lab at Rosalind Franklin University studies how stress and pain pathways interact in the brain. They used fiber photometry to monitor calcium fluorescence in the mPFC (medial prefrontal cortex) of mice traversing an elevated Zero maze while subject to inflammatory pain. They simultaneously monitored anxiety-like behavior by using video tracking to determine how much time the mice spent in exposed vs enclosed areas of the maze. They found that calcium fluorescence increased in male mice placed in the maze while subject to inflammatory pain, but there was no change in female mice under those conditions. Combined with other results, this finding suggests that sex-linked differences in the neural circuit between the locus coeruleus and mPFC are related to the differences in behavior and cognition displayed by male and female mice when subjected to inflammatory pain.

fiber photometry

FREE Fiber Photometry eBook

Amuza offers a FREE Wireless Fiber Photometry eBook. This ebook introduces topics and references critical for using fiber photometry during behavioral experiments

Cardenas A, Papadogiannis A, Dimitrov E. The role of medial prefrontal cortex projections to locus ceruleus in mediating the sex differences in behavior in mice with inflammatory pain. FASEB J. 2021 Jul;35(7):e21747.
https://pubmed.ncbi.nlm.nih.gov/34151467/

Animals: male and female mice
Sensor: GCaMP6f (calcium)
Vector: pAAV5.Syn.GCaMP6f, pAAV5.Syn.GCaMP6f.WPRE.SV40
Target Region: right mPFC (medial prefrontal cortex)
Coordinates: 1.8, ±0.4, and −2.2 mm in respect to bregma
Fiber: fiber core 400 μm NA 0.39, length 3 mm
Behavior test: Elevated O-maze
Fiber photometry data analysis: Amuza TeleFipho software
Model: Injection of complete Freund’s adjuvant (CFA) as a model for inflammatory pain.
Results: Inflammatory pain altered the calcium fluorescence signal from the mPFC of male mice placed on an elevated 0-maze, but did not alter the signal in female mice.

My lab has been using TeleFipho wireless photometric system for the past two years. The system is simple to use, durable and reliable. The practicality of TeleFipho allowed us to collect in vivo data about the neuronal activity of various limbic regions of the CNS during behavioral tests in mice.

Eugene Dimitrov MD, PhD

Assistant Professor, Department of Physiology and Biophysics Center for the Neurobiology of Stress Resilience and Psychiatric Disorders Rosalind Franklin University of Medicine and Science

Other mazes which require a mouse to pass through or a doorway or tunnel, such as the puzzle box maze, Light/Dark box, and many social interaction tests, can also present difficulties when using patch cords. Yunlei Yang’s lab used Amuza fiber photometry while mice explored a light/dark box to help characterize an anxiogenic circuit between septal OXTr neurons and the HDB, and identified a possible cause of OXT therapy side effects.

Huang, Tuanjie, Fangxia Guan, Julio Licinio, Ma-Li Wong, and Yunlei Yang. “Activation of septal OXTr neurons induces anxiety-but not depressive-like behaviors.” Molecular Psychiatry (2021): 1-10.
https://pubmed.ncbi.nlm.nih.gov/34489531/

Animals: Male and female C57BL/6 J and Oxtr-Cre mice.
Sensor: GCaMP6s (calcium)
Vectors: AAV1-hSyn1-GCaMP6s-P2A-nls-dTomato, AAV1-hSyn1-axon-GCaMP6s
Target Region: Fiber: vHPC (ventral hippocampus) Vector: lateral septum.

Fiber: fiber core 400 μm diameter, NA 0.39
Behavior test: light-dark box, Elevated Plus Maze
Fiber photometry data analysis: Amuza TeleFipho software
Results: Anxiogenic conditions activate the vHPC and vHPC projections to the lateral septum, as shown by increased calcium levels.

Problems with tethers also apply when using optogenetics and EEG, which is why we recommend using wireless optogenetics and EEG with freely moving animals.

5 Tips for Successful EEG and EMG Recording in Freely Moving Mice and Rats

5 Tips for Successful EEG and EMG Recording in Freely Moving Mice and Rats

Sleep research frequently relies on EEG (Electroencephalography) and EMG (electromyography) to discern between different sleep and wake states of the mice and rats used as models. The combination of EEG and EMG is powerful enough to discern not just between the different sleep and wake states, but also between the different stages within sleep and wake states.

In this article, we’ll show you how to reliably record EEG and EMG, with neural stimulation in untethered mice and rats.

EEG is also frequently used to record seizures in mouse and rat models of epilepsy, as well as for studying Huntington’s, Alzheimer’s, schizophrenia, migraine, and other disorders. Typically epidural EEG, aka Electrocorticography (ECoG), where the electrodes are placed directly on the cortex, is used.

1. Consider alternatives to tethers in EEG experiments

Equipment choice affects the success or failure of many EEG experiments. Typically, a tethered system is used, with implanted electrodes connected via a long cable to the recording system. Tethers are often the weak link in EEG experiments. Tethered mice become tangled, especially during seizures, severely limiting normal mice behavior. Slip rings, swivels, and balance arms can help prevent tangling, but mice do not create enough torque when they move to rotate many slip rings. Rats also chew on the cables, frequently damaging or destroying them. Complete disconnections also occur when animals pull on the cables, ending the recording and injuring the animals.

As an alternative to tethered animals, wireless EEG equipment helps reduce altering the normal actions of freely moving and behaving animals. Wireless EEG equipment also reduces the limitations of environments available for studying behavior.

 

2. Eliminate electrical noise and artifacts

Tethered EEG equipment often suffers from poor signal-to-noise ratios. Cables act as antennae, picking up electrical noise from AC power lines, motors, and other sources. This leads to artifacts in the data, complicating interpretation. Faraday cages can limit noise from external sources but become awkward and inconvenient when working with multiple animals. Faraday cages also do not protect against motion artifacts caused by the movement of the tether itself. Even connecting a preamplifier directly to the electrodes, so that amplified signals are sent through the tether, does not always eliminate AC and other noise from the system. A preamplifier can also make the system too heavy for use with mice. Additionally, multiple tethered systems in the same room can also lead to interference and noise.

3. Eliminate system limitations that affect your EEG experiment

The issues surrounding tethers have led to a shift to wireless EEG equipment. Wireless inductive telemeters addressed many of the problems of tethered systems, but bring with them other challenges.
Limits to Size and Type of Environment – Inductive systems require the animals to remain directly on top of a special charging pad or platform, limiting the size and type of environments which can be used during behavioral research.
Limits to Channel Bandwidth – Some telemetry systems also limit the bandwidth or number of channels available for EEG signals.
Limits to Sensitivity and Scale Up – Surprisingly, some wireless systems are still vulnerable to AC noise, possibly because of inadequate shielding. The number of frequencies available also limits wireless systems using radio to send data, capping the number of animals that can be monitored in one room.

As an alternative, researchers can use a lightweight data logger small enough to sit on the head of a mouse. The electrodes connect directly to the well-shielded data logger, preventing electrical noise from power lines.
Since data is stored onboard using a microSD card, there is less chance of signal interference and scaleup to more animals is unlimited.

The rechargeable data loggers are as small as 2g, allowing mice to behave normally during chronic recording sessions in any size cage or chamber.

 

4. Reduce electrode implant surgery time and complexity

PCB-based electrodes can make implantation surgery more complicated and time-consuming, leading to higher failure rates. A simple, flexible electrode design, such as the one shown below, makes for easy implantation.

The Amuza standard electrode set uses a universal 1.27 mm pitch pin and socket connector, making it easy to create your own electrode sets. Our standard electrode has two screws for EEG, two silver wires for EMG, and one screw for GND.

5. Look for universally compatible EEG data processing and analysis

As part of a research team, or in a collaborative setting, it is important to consider using EEG equipment that makes for seamless sharing of data. Proprietary data formats and readers can complicate sharing and processing your data. Incompatible systems can become an unexpected expense when you need one software license to run your EEG system and another for the PC in your office where you process the data.

Amuza’s EEG data analysis software combats these unplanned expenses in four ways:

  1. Amuza’s EEG data analysis software is included with the system free of charge
  2. Amuza’s EEG data analysis software can be installed on multiple computers without extra licensing fees.
  3. Through an intuitive platform, Amuza’s EEG data analysis software enables a quick overview of your data, as well as

    filtering and simple (FFT-based) alpha, theta, delta power calculation. It can also create hypnogram plots for sleep analysis. 

    4. Data from Amuza’s EEG data analysis software is also compatible with MatLab and Octave and can be exported to EDF or TXT. No dedicated reader or software is required.

Amuza’s EEG data analysis software combats these unplanned expenses in four ways:

  1. Amuza’s EEG data analysis software is included with the system free of charge
  2. Amuza’s EEG data analysis software can be installed on multiple computers without extra licensing fees.
  3. Through an intuitive platform, Amuza’s EEG data analysis software enables a quick overview of your data, as well as

filtering and simple (FFT-based) alpha, theta, delta power calculation. It can also create hypnogram plots for sleep analysis. 

4. Data from Amuza’s EEG data analysis software is also compatible with MatLab and Octave and can be exported to EDF or TXT. No dedicated reader or software is required.

There is no other system which meets my requirements for wireless operation, channel count, and usability with mice.

Yet-to-be-published ELG-2 User

Share your work! Tell us how you have struggled with tethered research animals in the comments below.

2021 Optimizations to Wireless Fiber Photometry and Wireless Optogenetics Products, Based on Customer Feedback

2021 Optimizations to Wireless Fiber Photometry and Wireless Optogenetics Products, Based on Customer Feedback

Amuza has spent the last year listening to researchers who use optogenetics and imaging techniques with mice and rats and learning from their experiences. As customers use our products their suggestions for how to optimize our products have been invaluable, which sent our engineers back to work. The results are six new upgrades, options, and improvements for our wireless products, making them easier to use, allowing experiments to run longer, and providing a better customer experience.

Updates to Wireless Neuroscience Products

1. Removable, Rechargeable Batteries to Keep Experiments Running

One of the first questions researchers ask us about our wireless neuroscience products is how long the batteries will last, since this determines how long the experiment can run before the headstage needs to be swapped for recharging. To date, Amuza wireless neuroscience systems have been powered by integrated rechargeable batteries. We have offered versions with larger batteries, but this does increase the weight.

Our wireless systems are now offered with removable rechargeable batteries in several sizes. The batteries connect using a simple plug: To continue your experiment, just swap the battery for a recharged one. Choose a standard battery for mice (total weight for TeleFipho, 3.3 g), or a larger battery (total weight 5 g) with 60% longer battery life for rats.

TeleFipho headstage (left) and removable batteries (center, right).

2. Wired, but better.

Not entirely an update to our wireless products, but still highly relevant for long experiments and experiments where switching the battery would disturb the animal. Amuza now offers a wired version of our fiber photometry system. Unlike fiber photometry using optical patch cords, the headstage still contains the light source and fluorescence detection system. For the tether: instead of an optical signal, power and the amplified data signal are sent through a slim electrical cable. Because of this, compared to other wired systems, the wired version of TeleFipho does not degrade the signal by passing it through multiple optical connections or a rotary joint on the way to the detector. The cable is also very flexible, and bending it will not introduce artifacts into the signal. Just let us know what type of plug we should use on the cable, and we can make certain it will be compatible with the commutator you choose, preventing the cable from twisting or tangling.

Wired TeleFipho

3. Superior protection from interference for cleaner data on fiber photometry

To optimize the data transmission of our wireless fiber photometry system, we improved the shielding and the antenna on the TeleFipho headstage. This cuts down on both electrical noise in the data, and also makes the radio communication more robust in environments where radio interference has been an issue. In addition to the shielding and antenna improvements, a new directional antenna for TeleFipho provides a great solution for radio interference in more complex environments. If you experienced signal dropouts with TeleFipho’s standard antenna, this antenna should completely eliminate them.

4. New fiber optic cannula sizes for fiber photometry and optogenetics

TeleFipho has now been tested with 200 μm (core) diameter fibers as well as 400 μm. Narrower fibers offer the benefit of causing less physical trauma, as well as being able to target smaller structures. But they collect much less light from the target region, so bright, well expressed fluorescent biosensors are required to get high-quality data. Many of our customers in Japan have now used 200-micron fibers with TeleFipho to detect calcium using newer versions of GCaMP and reported excellent results. We now offer fiber optic cannula with either 200 or 400-micron fibers, cut to the length you specify. The cannula can also be used with our optogenetics system. Please see our blog post on fluorescent biosensors for updated information on sensors used for fiber photometry.

5. Scaling up fiber photometry for more animals

At launch, TeleFipho had four separate radio channels, allowing up to four animals to be monitored in the same room. We added 4 more radio channels to TeleFipho, so now up to 8 systems can be used together simultaneously.

6. Headstage protection chamber prevents equipment damage

Group housing gives animals the opportunity to nibble on or dislodge headstages, plus some cages have wire lids and other surfaces which can catch head-mounted equipment. Our new protection chamber isolates the headstage from the environment but is easy to remove between experiments. We recommend using the protection chamber with rats, non-human primates (NHPs), and other larger animals.

For more information on customizing wireless neuroscience products for your experiments to obtain better data from your wireless system, visit our wireless neuroscience products page.

Which upgrade is the most helpful for your research? What other upgrades would be valuable to you? Leave a comment below.

Very Fast CRISPR (vfCRISPR) synchronizes and improves the accuracy of CRISPR cleavage using 365 nm light as a trigger.

Very Fast CRISPR (vfCRISPR) synchronizes and improves the accuracy of CRISPR cleavage using 365 nm light as a trigger.

Background:
CRISPR creates site-specific double-strand breaks (DSBs) in living cells. The system requires two components: a guide RNA (gRNA) to target a specific DNA sequence, and a CRISPR-associated endonuclease (Cas9 protein) to perform the actual cleavage. While the actual cleavage is quick, the initial steps take many hours: transfection of the cells, expression of the guide RNA and/or Cas9 protein, nuclear localization, target searching, and eventual formation of the gRNA-DNA-protein complex at the correct site. Because of this, the actual cleavage events are also spread out over a long period of time. Traditional CRISPR-Cas9 editing can also be inaccurate, with many DSBs happening off-target at locations far away from the intended site.

Incubator compatible 365 nm LED array light sources for 96 well and other multiwell plates are available from Amuza. Our LEDA led array light sources are designed for precise, reproducible illumination in optogenetics, photochemistry, and other applications.

How CRISPR becomes Very Fast: A 365 nm wavelength stoplight

The Johns Hopkins team, led by Profs. Taekjip Ha and Bin Wu created vfCRISPR by photocaging specific deoxythymidine residues of the guide RNA resulting in a caged guide RNA (cgRNA). This cage still allows the cgRNA and endonuclease to bind at the target DNA sequence, but the cages physically block the HNH portion of Cas9 from cleaving DNA. Illumination with 365 or 405 nm light releases thymidine from its cage, allowing DNA cleavage to proceed. To synchronize cleavage across an entire cell sample, the CRISPR components are first allowed to incubate and complete the slow localization and binding steps of the process. This ensures the cgRNA-DNA-protein complexes have already formed and are primed to begin cleavage before the ~30 second illumination period begins.

Benefits of vfCRISPR

  • Fast cleavage: Normally DNA cleavage by CRISPR-Cas9 is spread over many hours, as it takes time for the gRNA and Cas9 protein to enter the nucleus and bind to the correct DNA sequence. With vfCRISPR, 50% of the target sequences can be cleaved within 30 seconds of illumination.
  • Fast Repair. Ensuing DNA damage repair was faster and more concerted compared to repair after other methods of DNA damage. The team found that DNA damage response (DDR) proteins were recruited within 2 minutes of cleavage and repair was complete within 15 minutes.
  • Increased accuracy and precision. Both accuracy and precision of cleavage were improved, with fewer off-target strand breaks and shorter deletions of more consistent length. It was also possible to target just a single allele for cleavage. Allele-specific CRISPR is being used to create isogenic iPSC cell lines for both disease models and also for potential cell therapies.
  • Few artifacts. No observed phototoxic effects were observed in the cells after illumination.

The combination of control in both time and location makes vfCRISPR a likely tool for studying DNA damage repair. Frequently these studies have been performed using much less precise methods of damaging DNA, resulting in more variability in the observed kinetics of the ensuing DNA damage response. But with vfCRISPR, all of the damage is to the same gene at the same time, so the kinetics and position dependence of DNA repair can be studied more accurately.

The photochemistry:

vfCRISPR uses 6-nitropiperonyloxymethyl caged deoxythymidine (NPOM-caged-dT) to arrest cleavage. Several caged-dT residues are added to the distal end of each guide RNA. Located there they don’t interfere with binding to the targeted DNA sequence or binding of Cas9 to the DNA-RNA complex but do prevent DNA cleavage. Exposure of the caged dT residues to UV light begins a two-step process which results in the generation of the uncaged thymidine, allowing cleavage to proceed. The Johns Hopkins team found that 1.3 J/cm2 (~ 40 mW/cm2 for 30 seconds) of 365 nm light was sufficient to uncage cgRNAs.

NPOM-caged-dT modified RNA as well as phosphoramidites are available from Glen Research and Gene Link.

Liu, Y., Zou, R. S., He, S., Nihongaki, Y., Li, X., Razavi, S., … & Ha, T. (2020). Very fast CRISPR on demand. Science, 368(6496), 1265-1269.

Questions?

Microdialysis vs Fiber Photometry for Neurotransmitters

Microdialysis vs Fiber Photometry for Neurotransmitters

Microdialysis and Fiber Photometry are both powerful techniques for monitoring the concentrations
of neurotransmitters in vivo, but each technique has very different advantages. Amuza is the only company that provides both types of equipment to the neuroscience community, putting us in a unique position to help you determine when microdialysis or fiber photometry would be the best choice.

MicrodialysisFiber Photometry
Best for
  • Monitoring long-lasting changes
  • Absolute concentrations
  • Many analytes
  • Sub nanomolar analytes
  • Monitoring fast changes
  • Relative concentrations
  • One or two analytes
  • Targeting cell subtypes
SensitivityPicomolarNanomolar
Multiple analytesMany analytes can be measured in each sample with the little added difficultyData processing and experimental protocol are both more difficult
Types of analytesMost types of molecules.Limited by available fluorescent sensors
Type of measurementAbsolute concentration or % change relative to baseline% Change relative to baseline
Data analysisRelatively simpleOften quite complex
Sampling period~30 seconds to hoursMilliseconds
Sampling durationDaysDays, but difficult to monitor slow changes
TargetingBrain region/structureThe brain region, specific projection, cellular subtype, axon vs cell body
Animal movementTether requiredTether or wireless headstage

Multiple analytes

It is routine to measure many different neurotransmitters or amino acids in each microdialysis sample using HPLC chromatography and electrochemical detection. Samples can also be split and stored before analysis so that additional analytes can be added to the protocol at a later date. Commercially available assays also allow panels of multiple peptides and proteins to be measured in each sample.

Measuring multiple analytes simultaneously using fiber photometry is possible, but it requires expressing multiple fluorescent sensors at the target and the data analysis also becomes much more complex

Types of analytes

Microdialysis is extremely flexible and has been used to measure many types of analytes including amino acids, proteins, peptides, neurotransmitters, sugars, and more. If you can assay for it, you can probably sample for it using microdialysis.

Fiber photometry can now measure most of the principal neurotransmitters but is limited by the fluorescent sensors available. The number of sensors is increasing rapidly, but the options are still smaller than those available to microdialysis users.

Type of measurement

Microdialysis allows the monitoring of absolute concentrations or concentrations relative to basal levels in every sample.

Fiber photometry data yields a change in fluorescence data (∆F/F0). This correlates with changes in the concentration of the analyte relative to baseline but typically isn’t used to determine absolute concentrations.

Data Analysis

HPLC software packages already include all of the functions required to automate the processing of microdialysis data, allowing the monitoring of many analytes simultaneously over time. The analysis can be easily taught to new users. In contrast, fiber photometry data processing is still usually a complex operation, with most labs using multiple software packages and writing their own scripts to deconvolve and analyze multiple streams of data. (TeleFipho photometry data only uses fluorescence data from one wavelength, and is easier to process)

Sampling Period and Duration

While sample times can be as short as 30 seconds, microdialysis excels in measuring long-lasting changes in extracellular concentrations, and for determining absolute concentrations for baseline levels. It is quite routine to continuously sample for many hours or even days during a microdialysis experiment. Furthermore, the microdialysis probe can be removed and replaced with a dummy probe for days or weeks between sampling sessions.

Fiber photometry is at the opposite extreme: fluorescence is measured every 1 to 10 ms, allowing it to record events lasting less than a second. But drift, noise, and photobleaching during fiber photometry experiments complicate observation of slow changes over time.

Targeting

Both microdialysis and fiber photometry are used to target a discrete region or structure within the brain. Fiber photometry also allows retrograde or anterograde targeting, so that only specific projections are monitored. Viral vectors that only express the sensor in a specific cellular subtype or target either the axon or cell body can further narrow the scope of the events recorded.

Animal movement

Microdialysis and fiber photometry both typically require a tether during the experiment. TeleFipho is the exception and uses a wireless rechargeable headstage to gather and transmit fiber photometry data.

Do you have a question about microdialysis or fiber photometry?

GECIs and GEFIs: A Guide for Choosing Fluorescent biosensors for fiber photometry

GECIs and GEFIs: A Guide for Choosing Fluorescent biosensors for fiber photometry

Fiber Photometry is a rapidly advancing field, with biosensors for more analytes and with better sensitivity being announced almost every month. We would like to share information about sensors that should be compatible with fiber photometry when using excitation with blue (~480 nm) light and measuring green (~525 nm) fluorescence. This is the most commonly used wavelength pair and is offered with TeleFipho wireless fiber photometry.

We will update this guide as more information becomes available.

Overview of Fluorescent indicators: Structure and considerations for use.

Genetically encoded fluorescent indicators (GEFIs) are used in conjunction with fiber photometry to report on changes in concentrations of molecules in vivo in real-time.

Most fluorescent biosensors comprise a fluorescent protein yoked to an analyte binding protein, constructed so that binding of the analyte causes a dramatic increase in fluorescence.

Akerboom, Rivera, Guilbe, Malavé, Hernandez, Tian, Hires, Marvin, Looger, Schreiter ER / CC BY (https://creativecommons.org/licenses/by/3.0)

When used with fiber photometry in behaving animals, the sensors are usually introduced by injecting viral expression vectors. The virus is used to both express the sensor and to control its location: targeting sequences allow the researcher to choose a specific cellular subtype, and even the cellular localization, such as axon or soma, where the sensor will be expressed. Anterograde and retrograde localization can also target only a specific projection or circuit in a target region. Transgenic animals are also available for expressing some GEFIs.

Binding kinetics helps determine the range of concentrations the sensor will respond to, and its ability to report fast events. A sensor with high affinity (low Kd) and a long dissociation time can measure very low concentrations of a molecule, but this happens at the expense of being able to resolve more frequent events and a narrower useful range of concentrations. Fast dissociation improves time resolution, but sensitivity usually suffers.

The brightness of the sensor, partially expressed as the ratio of the increase in fluorescence when bound to the analyte compared to baseline (∆F/F or ∆F/F0), is the other major factor to consider. Brighter sensors can generate a useful signal when expressed at lower levels or when used with less illumination when compared to less bright sensors. They can also be used with narrower fibers. Some of our users are stepping down to 250 microns from 400-micron core diameter when using the latest generations of GCaMP type sensors for fiber photometry.

Most biosensors are already available from AddGene: some as plasmids, others as aliquots of ready-to-use viral vectors. The newest biosensors listed here can be sourced directly from the laboratories which invented them. We included the best source we could find, and the original publication describing the sensor in the table below.

Calcium

Calcium SensorsAffinity (Kd or EC50)dissociation
Kinetics (Mean life,
1/Koff)
∆F/F0 (% increase)Source for vector or plasmidReference
GCaMP6s147 nM1796 ms1680
Addgene
Chen, 2013
GCaMP6f375 nM400 ms1314
Addgene
Chen, 2013
jGCaMP7s68 nM1260 ms
Janelia
Dana, 2019
jGCaMP7f150 nM270 ms3100
Janelia
Dana, 2019
jGCaMP8f334 nM27 ms7880
Addgene
Janelia
jGCaMP8m108 nM55 ms4570
Addgene
Janelia
jGCaMP8s46 nM272 ms4950
Addgene
Janelia
GCaMP-X
Addgene
Yang, 2018

The GCaMP6 series of genetically encoded Ca2+ indicators (GECIs) are the most popular tools for examining action potentials and have been used extensively with TeleFipho photometry. GCaMP6f is optimized for fast decay kinetics, necessary for monitoring quick events, while GCaMP6s have higher sensitivity and slower decay kinetics. If you are starting a new project, consider the latest generation – jGCaMP7 – which offers higher sensitivity and a larger range of kinetics.

The jGCaMP7 series was introduced in 2019 as a collaborative effort between Loren Looger at Janelia and other research institutes. The jGCaMP7 GECIs have several-fold higher ∆F/F0 and a wider range of kinetics when compared to the earlier GCaMP6 sensors. Some GCaMP7 variants that will interest fiber photometry users include jGCaMP7s (highest sensitivity, but slower kinetics), and jGCaMP7f which has the fastest kinetics. We hope to have calcium data generated using jGCaMP7s and TeleFipho wireless fiber photometry soon.

The  jGCAMP8 series from The Looger Lab and the GENIE Project Team at HHMI Janelia was introduced in late 2020 and is the most recent set of GECIs available with improved sensitivity and speed. Compared to jGCAMP7f, the new series all have a faster rise time. 8f (fast) has a 4x faster rise time and a 2.5x faster decay time. 8m (medium) again has about a 4x faster rise time but is also 3.5x more sensitive. 8s (sensitive) is 2x more sensitive and 2x faster. A bonus of using these newer, brighter sensors for fiber photometry is that narrower fibers can be used. Some of our users are stepping down from 400 microns to 250-micron core diameter fibers, allowing for less traumatic surgeries while targeting smaller areas.

GCaMP-X The calmodulin GCaMP based calcium sensors have been shown to cause side effects during some in-vivo uses, such as interference with the function of L-type calcium channels, nuclear accumulation, and cytotoxicity. Changes largely addressed these issues to the design of GCaMP-X.

Dopamine

Dopamine is rapidly becoming the second most common target for imaging and photometry in neuroscience thanks to two sensors introduced in 2018, dLight and GRABDA. The intensity of illumination used with dLight and GRABDA is typically 20 – 30 μW, the same range as is used with GCaMP6.

Dopamine sensorsAffinity
(Kd or EC50)
dissociation
Kinetics
(residence time,
τ = 1/Koff)
∆F/F0
(% increase)
Source for vector or plasmidReference
dLight1.1330 nMNA230
Addgene
Patriarchi, 2018
dLight1.2770 nM90 ms340
Addgene
Patriarchi, 2018
dLight1.3b1680 nM930
Addgene
Patriarchi, 2018
GRABDA1m130 nM700 ms90
Addgene
Sun, 2018
GRABDA2m90 nMNA340
Yulong Li Lab
Sun, 2020
GRABDA1h10 nm2500 ms90
Addgene
Sun, 2018
GRABDA2h7 nMNA280
Yulong Li Lab
Sun, 2020

dLight1.1 and dLight1.2, developed by the Tian lab, have both been used extensively with fiber photometry, with settings similar to those used for GCaMP6.

GRABDA DA2M, DA2H

GRABDA (GPCR-Activation Based DA) was first introduced by Yulong Li’s lab in 2018 and has just been updated to increase Δf/f and increase the range of kinetics. The recent versions are DA2H (high affinity) and DA2M (medium affinity). Both GRABDA2m and GRABDA2H have already been used with fiber photometry, but so far results have only been communicated via preprints.

Norepinephrine and Serotonin

More from the Yulong Li lab, though as of yet their characterization is only available through preprints. GRABNE1m and GRAB5-HT1.0 have both already been used to measure norepinephrine and serotonin via fiber photometry in mice.

SensorsAnalyteAffinity
(Kd or EC50)
dissociation Kinetics
( τ = 1/Koff))
∆F/F0
(% increase)

Source for
vector or plasmid

Reference
GRABNE1hNorepinephrine83 nM2000 ms130
Yulong Li Lab
Feng, 2019
GRABNE1mNorepinephrine930 nM750 ms250
Yulong Li Lab
Feng, 2019
GRAB-5HT1.0Serotonin22 nM3.1 s280
Yulong Li Lab
Wan, 2020
iSeroSnFrSerotoninEC50 1.5 µm
Tian Lab
Unger, 2020

Biosensors for endo cannabinoids (GRABeCB), ATP, cholecystokinin (CCK), vasoactive intestinal peptide (VIP), somatostatin (SST), vasopressin/oxytocin, ghrelin, and orexin were also announced by the Li lab at Neuroscience 2019, and are still being validated. The best way to keep up with the Li lab may check #GRABSensors on Twitter!
More information on iSeroSnFr from the Tian lab should be available soon.

GABA

GABA SensorsAffinity (Kd or EC50dissociation
Kinetics
(τ = 1/Koff)
∆F/F0 (% increase)SourceReference
iGABASnFR9 μM250
Addgene
Marvin, 2019

Glutamate

There are two main sensor types available for monitoring glutamate: iGluSnFR and iGlu. The original iGluSnFR has slower kinetics, while iGluf (fast) and iGluu (ultrafast) are much faster. The new SF-iGluSnFR variants offer higher brightness and a range of different kinetics compared to the original.

Glutamate SensorsAffinity (Kd or EC50dissociation
Kinetics
(τ = 1/Koff)
>∆F/F0 (% increase)SourceReference
iGluSnFR4.9 μM92 ms100
Addgene
Marvin, 2018
iGluf137 μM2.1 ms
Addgene
Helassa, 2018
iGluu600 μM0.7
Addgene
Helassa, 2018

Acetylcholine

Acetylcholine SensorsAffinity (Kd or EC50dissociation
Kinetics
(τ = 1/Koff)
∆F/F0 (% increase)SourceReference
iACHSnFR1.3 µM1200
Addgene
Borden, 2020
ACh3.02 μM3.7s
Yulong Li Lab
Jing, 2019
ACh4.3
Yulong Li Lab

iACHSnFR is one of the most recent GEFIs created by the Loren Looger lab at Janelia, along with collaborators.

GACH and GRABACh3.0
While the initial version of the GRAB type acetylcholine indicator (GACH) was not sensitive enough for measuring physiological levels of ACh using fiber photometry (personal communication), the version described in a preprint from Dec. 2019 (ACh3.0) has been used successfully with fiber photometry. The Li lab is also supplying researchers with an even newer version, ACh4.3.

Other Analytes

Adenosine and ATP (Extracellular)

SensorsAnalyteAffinity (Kd or EC50dissociation
Kinetics
(τ = 1/Koff)
∆F/F0 (% increase)Source for vector or plasmidReference
GRABATP1.0ATPEC50 ~45 nM9 ms500-1000
Yulong Li Lab
Wu, 2021
iATPSnFR1ATPEC50 of ~50 nM190
Addgene
Lobas, 2019
GRABAdoAdenosine60 nM63 ms120
Yulong Li Lab
Peng, 2020

Adenosine
Peng et al. monitored adenosine in the mouse basal forebrain using fiber photometry and GRABAdo, also called Ado1.0

Please let us know if you have any corrections or additions to this list!

Telefipho Wireless Fiber Photometry

Telefipho Wireless Fiber Photometry

Telefipho Wireless Fiber Photometry

We would like to tell you about Amuza’s new wireless fiber photometry (TeleFipho). It’s our newest way to track calcium and neurotransmitters, and it works in real-time in freely moving animals. It’s easy to use, easy to process your data, and it’s ready to go right out of the box.

Fiber photometry is one of the newest tools available to neuroscientists who wish to correlate behavior with neural activity. It’s a powerful, ultra-fast technique used to measure calcium, neurotransmitters and other molecules in vivo in real-time. But until now the technique has required a connection – a fiber optic cable connecting the research animal to the rest of the optical hardware outside of the cage. This cable limits animals’ freedom of movement and social interactions. It is also fragile and can make your data noisier. This, in turn, can limit the design of your experiments.

Fiber Photometry Schematic

Telefipho Schematic

With Telefipho all of the optical hardware – light source, optical filters, and photodetector – are combined in a small headstage. The rechargeable headstage communicates by radio with a base station. Your animals can move through tunnels and doorways and interact freely during experiments. Telefipho can also improve video tracking: tracking software often confuses a swaying cable with a mouse that is still moving. This can complicate the scoring of freezing behavior during fear conditioning.

The headstage mounts directly on an FC size fiber optic cannula without any intervening cables or interconnects. This maximizes light transmission and minimizes noise and artifacts. This headstage weighs just 3 grams and has been tested with both mice and rats.

To use the system, an optical fiber is implanted with the tip placed at the brain region of interest. Blue light from an LED in the headstage is sent through the fiber to excite the fluorescent sensors expressed in the target region. The sensor molecules fluoresce in proportion to the concentration of the analytes, and some of the fluorescent light travels back through the fiber to be measured by a photodetector. A fluorescence filter cube, combining bandpass filters for excitation, a bandpass filter for emission, and dichroic mirrors are used to remove extraneous wavelengths and separate the light paths. The headstage transmits the data to the base station and then to your computer.

Since only a single narrow fiber is implanted inside the skull, the technique is much less invasive than imaging, especially for targets deep inside the brain.
The fluorescent signal is recorded and allows tracking of changes in analyte levels on a subsecond time scale.

TeleFipho data is also easy to work with. Removing the cable means removing the motion artifacts caused by rotary joints and long flexible waveguides, so you won’t have to process your data to correct for them.

TeleFipho software provides a real-time view of the data, allowing you to quickly optimize light levels and detection sensitivity. You can manually add timestamps and notes to the data, or you can connect your behavioral equipments’ outputs to TeleFipho and automatically align behavioral events with the fluorescence data.

You can also send the signal to your own recording equipment and process the data using your own software.

With the ever-increasing number of genetically encoded fluorescent indicators for molecules beyond calcium, such as dopamine, glutamate, acetylcholine, norepinephrine, endocannabinoids, and even cyclic monophosphates, fiber photometry is certain to become a versatile tool in your lab. This is doubly true since the vectors used to express the sensors are becoming increasingly precise at targeting specific cell types and circuits so that results are increasingly specific with less interference from off-target cells and molecules.

If you would like to learn more about TeleFipho fiber photometry, please contact Amuza.

Questions?

Optogenetics In Vitro: Illuminating Cells in Microplates

Optogenetics In Vitro: Illuminating Cells in Microplates

Teleopto LED illumination for 96 well plates from Amuza

Amuza’s Teleopto LEDA array: an incubator compatible illumination system for optogenetics, photobiology, and photochemistry in microplates.

Several years ago we found that while more and more work in optogenetics was being done in vitro, there was little in the way of equipment specifically designed to fill this role. Many labs built their own multiwell stimulation systems. These custom designs often performed brilliantly, but they took time to design and build and often made it more difficult to share protocols with other labs. We decided to provide a light source that is reproducible from well to well and from experiment to experiment, one that is ready to use in any lab.

The LEDA arrays are the result and now cell biologists, developmental biologists, chemists, neuroscientists, and others are using this array for optogenetics and to invent new techniques.

Original LEDA-B

Our most popular LED array is the LEDA-B: it is perfectly sized for a single 96 well plate. It has 96 LEDs with emission centered at a 470 nm wavelength. A lip surrounding the array locks your plate into place so that the wells stay centered over the LEDs. It can also be used with other microplates sharing the 96 well plate footprint. The cable is 2 m long, so only the array needs to be inside the incubator.

The LAD LED driver can be controlled manually, and you can vary the irradiance from zero up to roughly 1.5 mW/mm2, depending on wavelength. You can also use low voltage TTL pulses to trigger illumination. This way you can use a programmable pulse generator to control the LED array. Lowering the voltage of the pulse lowers the voltage sent to the LED array proportionately, allowing programmatic control of not just the timing but also the intensity of illumination. Our STO pulse generator is easy to program and use, but if you already have your own pulse generator or stimulator you should be able to use it with Teleopto. We now also offer programmable multichannel  LED drivers. These drivers do not require a pulse generator, they can be programmed via software on your PC.

Our LED arrays are available in many colors to match the ever-increasing variety of light-controlled tools available in life science, including:

White 

UV 365 nm

Violet 405 and 420 nm

Blue 450 and 470 nm

Green 525 nm

Yellow / orange 590 nm

Red 630, 660, & 740 nm

Infra-Red 850 & 940 nm

If you don’t see the wavelength you need listed on the Amuza website, please let us know, we may be able to find the correct LED for your application.

Our two-color arrays have two different color LEDs under each well. For example, a blue and yellow array allows you to work with step function (bistable) options: a blue pulse of light opens the channel and a yellow pulse closes it again.

Our LEDA4 and LEDA6 arrays allow you to control four or six different sections of the plate independently. This way you can trial different intensities or pulse trains simultaneously, saving your lab time and space when developing new protocols.

We also make these arrays in different sizes so that you can illuminate multiple plates at once, or change the placement of the LEDs to match the format of different well plates. These arrays are also perfect for illuminating Petri dishes, tissue culture bottles, flasks, and other vessels.

Our LED arrays are being used for a wide range of applications, including Cry2 based biomolecular condensates, developmental biology, Stem cell differentiation, Cardiac optogenetics, receptor activation, and protein expression, oncology, ophthalmology, development of novel opsins and other optogenetic tools, photochemistry, and neuroscience.

Questions?

Wireless Optogenetics Basics

Wireless Optogenetics Basics

Teleopto Wireless Optogenetics System Introduction

Introduction to Teleopto Wireless Optogenetic stimulation of mice, rats, and other small animals during behavioral testing.

Teleopto is a turnkey solution for optogenetic stimulation of mice, rats, and other small animals during behavioral experiments. Most systems use a stationary laser or LED and a long fiber optic cable for stimulation, limiting the ability of your animals to move during behavioral experiments. But since Teleopto is wireless, your research animals have complete freedom of movement and can move through doorways, tunnels, mazes, and even large open field environments.

The Teleopto system consists of remote control, wireless receiver headstages, and LED fiber-optic implants. The whole system is ready to use right out of the box. The remote sends an infrared signal to the receivers to turn the LEDs on and off. The receivers weigh as little as one gram and can be recharged between experiments. The implants are made to order with many options for light colors, single or bilateral fibers, fiber length, and fiber diameter.  Two-color implants are available for use with bistable opsins or stimulation and inhibition at the same site.

You can adjust the light power with a screwdriver. For two-channel receivers, the power for each channel is adjusted independently. Using our LPM light meter to measure the output lets you ensure that each mouse or rat in a group receives the same irradiance at the target.

Between experiments, receivers are recharged. Battery life depends on how frequently and how intensely you stimulate your mice or rats. We offer larger receivers with larger batteries for use with rats and smaller receivers for use with mice. It’s easy to switch receivers during an experiment, so chronic experiments lasting many days are possible.

Teleopto can be controlled manually, but normally a programmable pulse generator is used to send pulse trains. Our pulse generator is easy to program and use, but if you already have one it will probably be compatible with Teleopto

The pulse generator can, in turn, be triggered by behavioral events. A nose poke, lever, video tracking system, or other equipment can easily be used to trigger the start or stop of a pulse train. Amuza can work with you to make certain your behavioral equipment is compatible with Teleopto.

Teleopto is great for scaleup: a single IR emitter can control multiple receivers on multiple mice or rats. With additional emitters, one remote can be used with multiple operant conditioning chambers or cages. Additional emitters can also make certain there are no dead spots in a maze or other complex environment. The emitters have a range of 1-2 meters depending on lighting, but we also have high power IR emitters for use in large open field environments.

Questions?

How to Choose the Best Microdialysis Probe for Your Project

How to Choose the Best Microdialysis Probe for Your Project

Microdialysis Probes How to Choose the Best One

There are many types of microdialysis probes available – some labs even make their own microdialysis probes. This guide can help you choose the right microdialysis probe for your next project. To learn about how microdialysis works and how it can advance your research goals, please start with our overview: What Is Microdialysis?

If you will be doing chronic microdialysis to measure dopamine, serotonin, acetylcholine, amino acids or other small molecules in mouse and rat brains, then, our FZ and CX probes are the best choice. These offer industry-leading recovery rates (the ratio of the concentration of an analyte in a recovered sample to the concentration of the analyte in the area surrounding the microdialysis membrane) for small molecules.

When you are working with mice, we recommend lightweight CX-i probes as your first choice. The CX’s narrow-body allows for bilateral microdialysis, even if your targets are close to the midline. The CX probe snaps into its guide cannula, so it is quick to insert and remove.

For rats and for longer experiments with mice we recommend our newly released FZ probes. The FZ probe is secured in the AG guide cannula by a cap nut (AC-5), locking it into place. This way rats and cannot dislodge the probe by scratching at it. The FZ probe is very similar to our outgoing AZ probe: we kept the best parts of the AZ probe and then made it both easier to use and more robust for use with more active adult rats. The FZ probe uses the same guide cannula and accessories as the AZ probe.

The FZ probe is also offered with a microinjection option: a microinjection cannula (orange) is attached to the MI-AZ probe so that drugs, antibodies, and other molecules can be delivered to the microdialysis site at a specific time(s).

The FZ, MI-AZ, and CX probes are mainly used for chronic microdialysis and use guide cannulae to accurately position the probe during implantation and protect the probe during the experiment. The guide cannula can be surgically implanted well before your actual microdialysis experiment and allows you to quickly implant the probe itself just before beginning an experiment. A dummy probe can be used instead of a microdialysis probe during recovery, conditioning, or while waiting for an injected vector to begin expressing proteins. We recommend using each microdialysis probe only once, as microdialysis membranes may become fouled if used repeatedly. When this happens, the recovery rate of the probe may drop and alter the results of your experiment.

DZ probe

 

For acute microdialysis experiments, where the mouse or rat remains on the stereotaxic frame during the entirety of the experiment, we have the DZ probe. This is our smallest probe, and it is intended to be directly implanted without a guide cannula. While we recommend it for acute experiments, The DZ probe can be used for shorter experiments with freely moving mice.

Researchers use our AtmosLM™ large molecule microdialysis probes to measure levels of peptides, proteins, antibodies, and even ApoE (Apolipoprotein E) particles. These probes have membranes with a molecular weight cut off of one million daltons. The AtmosLM PEP probes are superior to other push-pull probes because we add a vent to equalize pressure between the lumen of the probe and ambient conditions. This stops convection – the bulk transfer of water and solutes being driven across the membrane by pressure – and greatly enhances the accuracy of sampling. We offer these probes with polyethylene – uncharged – and polysulfone – charged membranes, to match your analyte.

If you will need to do MRI or PET imaging studies with the guide cannula and probe in place, we offer C-I probes and guide cannulae. The C-I series are metal-free and won’t interfere with imaging.

OP Probe

TP Probe

All of the probes discussed above were designed for use in the brain, but we also have probes for peripheral tissues. The OP linear probe is designed for dermal use and has a needle at one end to make it easy to implant, and the TP flexible concentric probe can be used in blood vessels and the GI tract.

Many of our probes can be made with different membrane materials. If you would prefer a polyethersulfone (PES) or polyacrylonitrile (PAN) membrane instead of cuprophane (artificial cellulose), please ask us. All of our probes, stereotaxic adapters, and accessories are made to your custom size requirements in just two weeks – even for the smallest orders. We also maintain a large inventory of FZ, CX-i, and PEP probes ready to ship the day you place your order.

If you are planning a new project or having trouble with your current one, please give us a call and we can help you troubleshoot. Eicom has been manufacturing microdialysis probes and equipment for 30 years and we have an integrated support/sales team so you can rely on our expertise.

Questions?

Eicom HPLC-ECD used to study GABA’s role in Diabetes

Eicom HPLC-ECD used to study GABA’s role in Diabetes

The Phelps lab at the University of Florida used the Eicom HTEC-500 HPLC-ECD to determine the time-resolved secretion of GABA, taurine, and other amino acids from pancreatic islet beta cells. This helped them show:

  • GABA in human beta cells is mostly stored in the cytosol, not in vesicles.
  • An ion channel (VRAC) handles GABA secretion from beta cells.
  • Pulsatile release of GABA synchronizes and regulates the secretion of insulin from islets.
  • The depletion of GABA reserves in beta cells correlates with both type 1 and type 2 diabetes in human patients.

Roles of GABA

GABA, short for gamma-aminobutyric acid, has many roles in biology. It is best known as an inhibitory neurotransmitter in the nervous system. In fact, impaired clearance of GABA in the amygdala may contribute to alcoholism. GABA also acts to inhibit immune responses and regulates the secretion of hormones in the pancreas.

How GABA, Insulin, and Glucagon are related to diabetes.

Insulin is secreted in response to high blood sugar levels. It directs cells throughout the body to take up glucose from the blood, thus lowering blood sugar levels. As insulin and blood sugar levels drop, glucagon is secreted. Glucagon directs the liver to convert glycogen into glucose and release it. Bringing blood sugar levels back up and balancing the action of insulin. Beta cells secrete both of these hormones in pancreatic islets. The secretion of both of these hormones is regulated in part by GABA, which is also secreted by beta cells. In type 1 diabetes an autoimmune response attacks and destroys beta cells. But this immune response can be damped by the GABA secreted by beta cells. Administering GABA as a drug not only protects beta cells, it can also cause other pancreatic islet cells (alpha cells) to convert into beta cells. Helping to restore the beta-cell population. Because of this protective role, GABA is already being studied as a treatment for diabetes by the Swedish biotech Diamyd. With clinical trials led by Prof. Kenneth McCormick at the University of Alabama.

How is GABA secreted in the pancreas?

In the brain, secretory vesicles are responsible for storing and then releasing GABA from neurons. This same mechanism was long thought to be at work in the pancreas as well. Yet, the GABA transporter proteins necessary for this mechanism remained elusive in beta cells. Leaving a gap in the understanding of how GABA release occurs. Prof. Edward Phelps previously observed that instead of being held in vesicles, the bulk of GABA is loose within the cytosol of beta cells. This meant that another mechanism could be in play. He and his student Walker Hagan hypothesized that an ion channel, specifically VRAC (Volume Regulatory Anion Channel), could be responsible.

“There is a channel between the interior of the beta cell and the extracellular space, which we thought was worth investigating,” Phelps said. “The volume regulatory anion channel (VRAC) is known for another purpose. It is used to help cells maintain their shape by keeping the osmotic pressure inside and outside the cell in equilibrium. When this balance is disturbed and the cell shape changes, small organic chemicals known as osmolytes are expelled from the cell via the VRAC channel to help the cell regain its shape. When we artificially opened this channel in beta cells using low saline, we found that this channel also transports GABA.”

Changes in cell volume had already been implicated as a trigger for insulin secretion. Further supporting the possibility that VRAC channels could be the principal mechanism for GABA secretion.

Measuring GABA secretion in the pancreas

The Phelps lab used their HTEC-500 HPLC-ECD system to measure GABA and other amino acids in three phases of this project.

First, they confirmed that human pancreatic islets from both type 1 and type 2 diabetic donors are deficient in GABA when compared to those of non-diabetics. Then they measured GABA in perifusate from islets cultured in a Biorep perifusion system. Perifusion washes media though a small group of islets and allows recovery of the perfusate as time-resolved fractions. This way conditions can be varied and resulting changes in the secreted amino acids and hormones can be monitored over time. By varying the media washing through the islets, The Phelps lab was able to confirm that hypotonic media triggers the release of GABA from beta cells. Consistent with VRAC being responsible for secretion. Perifusion also allowed them to study the time-resolved relationship between GABA release and insulin secretion.

Finally, they created a knockout strain of mice unable to express the VRAC channel in beta cells: (βc-LRRC8A−/−). The beta cells in these mice are missing one of the subunits (LRRC8A) of VRAC, and no longer secreted GABA in response to osmotic stress. This lent further support to the role of VRAC in GABA release.

 

Representative HPLC chromatograms from the supernatant of a culture of 200 pancreatic islets. Hypotonic media opens VRAC channels as the cell attempts to relieve osmotic stress. Allowing the release of both GABA and taurine from islets into the supernatant (blue). VRAC channels remain closed in Isotonic media (gray). Resulting in lower concentrations of GABA and taurine in the supernatant. Samples were injected onto an Eicom HTEC-500 equipped with an FA-3ODS column, used for separating GABA, Glu, and other amino acids. An AS-700 autosampler was used to automate both derivatization and subsequent injection of the samples. Data courtesy of the Phelps lab.

 

Levels of GABA and Taurine released into the supernatant of islets suspended in low saline (hypotonic) and isotonic (3G) media. Figure courtesy of the Phelps lab.