GRAB Sensors - Tools for Precisely Measuring Neurochemical Dynamics in vivo

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).

	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)	NE	-	Green	Human a2A receptor	EC50~930nM Medium affinity	~230%	τon ~72ms， τoff~680ms	No coupling	[6]
	NE1h(NE2.2)	NE	-	Green	Human a2A receptor	EC50~83nM High affinity	~130%	τon ~36ms， τoff~1890ms	No coupling	[6]
	NEmut	NE	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.

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

Browse All GRAB Sensors

Sensors available include those for acetylcholine (ACh), dopamine (DA), 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 vaso-pressin (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.

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 for Rapid and Specific In Vivo Detection of NE." Neuron 102(4): 745-761 e748.
[7] Sun, F., et al. (2018). "A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice." Cell 174(2): 481-496 e419.
[8] Jing, M., et al. (2018). "A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies." Nat Biotechnol 36(8): 726-737.