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GRAB Sensors - Tools for Precisely Measuring Neurochemical Dynamics in vivo



1. Development Principle of GRAB Sensors

Through the efforts the Yulong Li lab in Peking University, many genetically-modified fluorescent sensors delivered by AAVs for visualizing specific neurochemicals were developed by coupling neurochemical-sensing G protein coupled receptors (GPCRs) with a circularly permutated fluorescent protein (cpFP). When neurochemicals bind to their specific GPCRs, the conformation change in these GPCRs gives rise to a change in fluorescence of cpFPs, which can be detected by fluorescence imaging (Fig 1). These sensors are named as GRAB (GPCR Activation-Based) sensors, which can be used to in vivo monitor the dynamic changes of these neurochemicals with high spatiotemporal resolutions.





Fig 1: Schematic drawing shows the principle of the green and red GRAB sensors. Upon binding its ligand, the conformational change in the GPCR is sensed and reported by cpGFP or cpRFP (modified based on reference 4).




2. Properties of Selected Published Sensor


Sensor

Neurochemical Measured

Version

Color

Scaffold

Affinity

Peak  Response (∆F/F0)

Kinetics

Coupling to Downstream Signaling Pathway

Reference

Ach2.0

Acetylcholine

First generation

Green

Human M3 receptor

EC50~1uM

~90%

τon ~200ms, τoff~ 800ms

Weak coupling

[8]

Ach3.0

Acetylcholine

Second generation

Green

Human M3 receptor

EC50~2uM

~280%

τon ~112ms, τoff~ 580ms

Negligible coupling

[3]

Ach3.0-mut

Acetylcholine

Second generation control

Green

Human M3 receptor

EC50~0uM
(W200A mutation)

~1.8%

-

-

[3]

DA1m

Dopamine

First generation

Green

Human D2 receptor

EC50~130nM
Medium affinity

~90%

τon~60ms, τoff~700ms

Negligible coupling

[7]

DA1h

Dopamine

First generation

Green

Human D2 receptor

EC50~10nM
High affinity

~90%

τon~140ms, τoff~2500ms

Negligible coupling

[7]

DAmut (1st)

Dopamine

First generation
control

Green

Human D2 receptor

EC50~0uM
C118A & S193N mutations

No effect

-

-

[7]

DA2m(DA4.4)

Dopamine

Second generation

Green

Human D2 receptor

EC50~90nM
Medium affinity

~340%

τon ~40ms,
τoff~1300ms

Minimal coupling

[2]

DA2h(DA4.3)

Dopamine

Second generation

Green

Human D2 receptor

EC50~7nM
High affinity

~280%

τon ~50ms,
τoff~ 7300ms

Minimal coupling

[2]

DAmut(2nd)

Dopamine

Second generation
control

Green

Human D2 receptor

EC50~0uM
C1183.36A & S1935.42N mutations

No effect

-

- [2]

rDA1m (rDA2.5m)

Dopamine

-

Red

Human D2 receptor

EC50~95nM
medium-affinity

~150%

τon ~80ms,
τoff~ 770ms

Minimal coupling

[2]

rDA1h (rDA2.5h)

Dopamine

-

Red

Human D2 receptor

EC50~4nM
high-affinity

~100%

τon ~60ms,
τoff~ 2150ms

Minimal coupling

[2]

rDAmut (rDA2.5mut)

Dopamine

Control

Red

Human D2 receptor

EC50~0uM
C1183.36A & S1935.42N mutations

No effect

-

-

[2]

NE1m(NE2.1)

Norepinephrine

-

Green

Human a2A receptor

EC50~930nM
Medium affinity

~230%

τon ~72ms,
τoff~680ms

No coupling

[6]

NE1h(NE2.2)

Norepinephrine

-

Green

Human a2A receptor

EC50~83nM
High affinity

~130%

τon ~36ms,
τoff~1890ms

No coupling

[6]

NEmut

Norepinephrine

Control

Green

Human a2A receptor

EC50~0uM
S5.46A mutation

No effect

-

-

[6]

Ado1.0

Adenosine

-

Green

Human A2A receptor

EC50~60nM

~130%

τon~36ms,
τoff~1890ms

Negligible coupling

[4]

Ado1.0mut

Adenosine

Control

Green

Human A2A receptor

EC50~0uM
F168A mutation

No effect

-

-

[4]

5-HT1.0

Serotonin

-

Green

Human 5-HT2C receptor

EC50~22nM

~250%

τon~200ms,
τoff~3100ms

No coupling

[1]

5-HTmut

Serotonin

Control

Green

Human 5-HT2C receptor

EC50~0uM
D1343.32Q mutation

No effect

-

-

[1]

Please note that most of the properties mentioned in the above are from measurements in vitro in cultured cells expressing the GRAB sensors.



3. How to Order?

WZ Biosciences is the sole distributor of GRAB sensors in AAV viruses for in vivo monitoring of neurochemicals (Fig 2).


Browse All GRAB Sensors


WZ Biosciences, GRAB Sensors


Sensors available include those for acetylcholine (ACh), dopamine (DA), norepinephrine (NE), Serotonin (5-HT), adenosine (Ado), adenosine triphosphate (ATP), vasoactive intestinal peptide (VIP), cholecystokinin (CCK), endocannabinoid (eCB), neuropeptide Y (NPY), corticotropin-releasing factor (CRF), arginine vasopressin (AVP), histamine (His), somatostatin (SST), melatonin (MT), anandamide also known as N-arachidonoylethanolamine (AEA), oxytocin (OXT), Histamine (HA) and so on. Most of the GRAB sensors also have Cre-dependent and mutant versions. Some are also available in both cpGFP and cpRFP formats. More GRAB sensors are under development.

For all the published GRAB sensors, researchers can directly order from WZ Biosciences with discounted price (subsidized by the Li lab). For all the unpublished GRAB sensors, WZ Biosciences can help coordinate the purchase from the Li lab. Researchers can also directly communicate with the Li lab at yulonglilab2018@gmail.com.



4. FAQs

1) What is the suggested amount of viruses for injection?

Depending on the titer of each lot, we recommend 200-400nl per site of the non-diluted viruses.

2) Whether GRAB sensors will work in rats as they are in mice?

The GRAB sensors work well in rats. According to the published literature, these sensors can be used to detect neurotransmitter dynamics in many organisms, including Drosophila, zebrafish, mice and zebra finches.

3) Do you have the GRAB sensor AAVs with a ubiquitous promoter or other tissue-specific promoter?

Please refer to the list of tissue-specific promoters below to choose your interested promoter. Please contact us if you do not find the promoter of interest.


Tissue

Promoter Name

Size

Description

Aplication

CNS hSyn 471bp Human synapsin I promoter Specific in neuron
CamKIIa 1.2kb Mouse α-calcium-calmodulin dependent kinase II promoter Specific in excitatory neurons in the neocortex and hippocampus
c-fos 1.7kb mouse c-fos gene promoter Specific in excitatory neurons
Mecp2 230bp mouse methyl CpG binding protein 2 promoter Specific in neuron, short
NSE 1.3kb Mouse enolase promoter Specific in neuron
Somatostat(SST) 1.2kb Human somatostatin I gene Specific in GABAergic Inhibitory neuron subtype (SST)
TH 2.5kb Rat tyrosine hydroxylase gene promoter Specific in dopaminergic neurons
GFAP 2.0kb Human glial fibrillary acidic protein promoter Specific in astrocyte
GFAP104 845bp Human glial fibrillary acidic protein promoter Specific in astrocyte
GfaABC1D(truncated GFAP) 681bp Human glial fibrillary acidic protein promoter Specific in astrocyte
ALDH1L1 1.3kb Human aldehyde dehydrogenase 1 family member L1 promoter Specific in astrocyte in thalamus
MBP 1.3kb Human myelin basic protein promoter Specific in oligodendrocyte
Liver ALB 2.4kb Mouse albumin promoter Specific in liver
TBG 460bp Human thyroxine binding globulin promoter Specific in liver
ApoEHCR-hAAT 1.3kb Chimeric promoter of the hepatocyte control region (HCR) from the APOE gene and the human α1-antitrypsin promoter (hAAT) Specific in liver
Heart aMHC 0.4kb Mouse myosin heavy chain alpha promoter Specific in cardiomyocyte
cTNT+intron 0.7kb Chicken cardiac troponin T promoter Specific in cardiomyocyte
Eye Rep65 0.7kb Mouse retinal pigment epithelium 65 promoter Specific in retinal pigment epithelium(RPE)
VMD2 promoter 0.65bp Human vitelliform macular dystrophy-2 promoter Specific in retinal pigment epithelium(RPE)
Pancreas Insulin 0.85kb mouse insulin promoter Specific in pancreatic β cells
PDX1 2.7kb Mouse pancreatic and duodenal homeobox 1 promoter Specific in pancreatic β cells
Blood vessel SM22a 0.45kb Mouse SM22 alpha promoter Specific in vascular smooth muscle cell
ICAM2 0.15kb Human Intercellular Adhesion Molecule 2 promoter Specific in endothelial cell
CD68 0.7kb human CD68 promoter Specific in monocytes and macrophages
F4/80 1.2kb mouse F4/80 gene promoter Specific in macrophages
Muscle MCK 1.3kb mouse muscle creatine kinase gene promoter Specific in muscle
3×enhancer McK 728bp modified mouse muscle creatine kinase gene promoter Specific in muscle
Kidney NPHS1 1.2kb mouse Nephrin gene promoter Specific in kidney

4) How much viruses are in one vial?
We provide the GRAB sensor AAVs in 50ul aliquots.
5) There are many AAV serotypes. Have you packaged any of GRAB sensors with other serotypes, such as a retrograde AAV serotype?
For most of GRAB sensors, we package them in the AAV9 serotype. If the retrograde serotype is required, we offer custom packaging services.


5. Research Background
One of the main focuses of neuroscience studies at cellular and molecular levels is to understand how neurons are connected and communicate with each other. Communication between neurons at the synapse depends on chemical transmissions. There are two types of chemical messenger at the synapse, neurotransmitters and neuromodulators. Neurotransmitters diffuse across the synaptic cleft to bind to ligand-gated ion channels on the postsynaptic cells, while neuromodulators bind to presynaptic and postsynaptic GPCRs. Upon the binding of neurotransmitters such as glutamate and GABA, ligand-gated ion channels rapidly depolarize or hyperpolarize the postsynaptic neurons and directly regulate the activity of these neurons. For most neuromodulators, theirs binding to GPCRs cascades serial events within the presynaptic and postsynaptic neurons and generate slow but long-term changes in these neurons.
To manipulate and monitor the communication between neurons, various methods have been established and many tools (e.g. delivery by AAVs) have been developed. Nowadays, optogenetics and chemical genetics tools are used to directly control the generation of presynaptic action potentials. Calcium indicators are used to monitor the signal propagation within the postsynaptic neurons. However, the techniques to monitor the releasing of neurotransmitters and neuromodulators with high sensitivity, specificity and precise spatial resolution are not well developed.


6. References
1.Wan, J., et al. (2021). "A genetically encoded sensor for measuring serotonin dynamics." Nat Neurosci.
2.Sun, F., et al. (2020). "Next-generation GRAB sensors for monitoring dopaminergic activity in vivo." Nat Methods 17(11): 1156-1166.
3.Jing, M., et al. (2020). "An optimized acetylcholine sensor for monitoring in vivo cholinergic activity." Nat Methods 17(11): 1139-1146.
4.Peng, W., et al. (2020). "Regulation of sleep homeostasis mediator adenosine by basal forebrain glutamatergic neurons." Science 369(6508).
5.Jing, M., et al. (2019). "G-protein-coupled receptor-based sensors for imaging neurochemicals with high sensitivity and specificity." J Neurochem 151(3): 279-288.
6.Feng, J., et al. (2019). "A Genetically Encoded Fluorescent Sensor