|Li Research Lab|
FAQ of GRAB sensors
Q1: What GRAB sensors are currently available?
A1: We've developed a wide range of neurotransmitter sensors, which vary in their apparent affinity, color and brightness. For now, our lab has published the first generation green fluorescent sensors for dopamine(GRABDA1h & GRABDA1m, Sun et al., 2018, Cell), acetylcholine(GACh1.0 & GACh2.0, Jing et al., 2018, Nature Biotechnology)and norepinephrine (GRABNE1h & GRABNE1m, Feng et al., 2019, Neuron), and also spectra-separated red dopamine sensors (rGRABDA1m & rGRABDA1h, Sun et al., 2020, Nature method (accepted)). We are continuously making endeavors to develop next generation GRAB sensors with better signal to noise ratio and more GRAB sensors for detecting other molecules.
Q2: What do the abbreviation h/m/l/mut mean, and what about the number?
A2: h/m/l is the abbreviation of high/median/low, which indicates the sensors' apparent affinity towards transmitter. Most of the GRAB sensors have transmitter-insensitive mutant for comparison with GRAB sensors to check signal specificity. The number represents the version of sensor, the higher version, the better performance.
Q3: How to use GRAB sensors?
A3: GRAB sensors are genetically encoded sensors for neurotransmitters, similar to other genetically encoded sensors (e.g. GCaMP). They could be expressed in specific cell-types and are able to detect dynamics of extracellular neurotransmitters in cultured cell, brain slice and living animal (e.g. fly, zebrafish, mice, rat, monkey etc.). For the aspect of instruments, GRAB sensors could perform well in epifluorescence microscopy, confocal microscopy, 2P microscopy and fiber photometry recording.
Q4: How to access GRAB sensors?
A4: For published GRAB sensors, plasmids are available at Addgene and the viruses can be purchased from companies (WZbioscience (USA), BrainVTA (China) and OBiO (China)). For getting access to other unpublished sensors, please contact yulonglilab2018(a)gmail.com for consent and help. Click here for the list of plasmids and viruses.
Q5: Could you share the list of viruses and transgenic flies of GRAB sensor?
A5: Click here for the list of viruses (information including virus type, serotype, promotor, version of sensor, publication etc.). The GRAB sensor transgenic flies are also included in the list, contact us by email to order these materials. Email address: firstname.lastname@example.org.
Q6: Can the sensors be expressed in vivo for a long-term period?
A6: Yes. We can record good signals from GRAB sensor after expression for 3 to 6 months in mice brain by AAV infection.
Q7: Can GRAB sensors detect excitability of neurons?
A7: No. The change of fluorescent intensity only indicates the dynamics of corresponding neurotransmitter, which has no direct correlation with excitability of neurons. Other approach is needed for detection of neuron excitability.
Q8: Is there any downstream G-protein/β-arrestin coupling for GRAB sensors?
A8: The tested GRAB sensors show minimal coupling with G protein and there is no internalization of GRAB sensors induced by coupling with β-arrestin.
Q9: Does the expression of GRAB sensors have specific effect on neurons?
A9: The tested GRAB sensors have no/ very little specific effect on neurons. For example: 1) Simultaneous patch-clamp recordings showed that the resting membrane potential, input resistance, membrane time constant and average spiking frequency of GACh2.0-expressing CA1 neurons have no significant difference comparing with nearby control non-expressing neurons. Moreover, AMPA, NMDA, and GABAergic responses, as well as paired pulse facilitation of AMPA responses in GACh2.0-expressing neurons, remained unchanged. Moreover, chronic AAV expression of GACh2.0 in the dentate gyrus of hippocampus did not affect the Cal590 fluorescence responses elicited by high K+. We made acute cortical slices after chronic in vivo lentiviral expression of GACh2.0 in barrel cortical L5 pyramidal neurons in mice. Simultaneous patch-clamp recordings showed that GACh2.0-expressing and control non-expressing L5 pyramidal neurons displayed the same resting membrane potential, input resistance, membrane time constant, and average spiking frequency, as well as AMPA, NMDA, and GABAergic responses, and paired pulse facilitation of AMPA responses (Jing et al., 2018, Nature Biotechnology). 2) The experiment on brain slices in hippocampus in mice and on larvae of zebrafish showed that the expression of NE1m had no effect on calcium signal (Feng et al., 2019, Neuron). We co-expressed 5-HT1.0 and jRCaMP1a in fly, and observed no change in calcium signal compare to the non-expressing fly (Wan et al., 2020, bioRxiv).
Q10: Can sensors be used on patients?
A10: No. The GRAB sensors can be used on cultured cells, tissues, organoid and experimental animals, but not in clinical.