The complex function in the brain is inseparable from the efficient and specific information exchange and integration between neurons. Neurotransmitters (such as dopamine, acetylcholine, and norepinephrine) are key biological small molecules and participate in the process of information transmission mediated by chemical synapses between neurons and are involved in a variety of physiological functions including development, information perception, learning/memory, emotion, motor regulation and higher-level cognitive behavior in the brain. Malfunction of neurotransmitters and receptors will cause devastating diseases, such as Parkinson's disease, Alzheimer's disease, addiction, and epilepsy. Therefore, the study of neurotransmitters is essential for understanding the mechanism of brain function.

Existing Tools for Neurotransmitter Imaging & Their Limitations

However, precise measurements of the spatial and temporal patterns of release of neurotransmitters, specifically dopamine (DA), during complex behaviors are lacking, due in large part to the limitations of existing methods for DA detection. Intracerebral microdialysis has long been the gold standard for quantitative measurements of extracellular DA concentration. However, its relatively slow sampling rate (> 1 min between sampling, typically ~10 mins) (Chefer et al., 2009) is not well suited to detect DA dynamic changes during complex and rapidly evolving behaviors.

Fast-scan cyclic voltammetry (FSCV) is an electrochemical method that can measure changes in extracellular DA concentrations with 10 ms temporal resolution and ~1 nM sensitivity (Robinson et al., 2008). However, FSCV requires substrate oxidization for signal detection, therefore it is not readily able to distinguish DA from other structurally similar neurotransmitters, such as norepinephrine (NE) (Robinson et al., 2003). Moreover, both microdialysis and FSCV require implantation of a relatively large probe (approximately 70–300 μm in diameter) into brain tissue, which limits the ability to achieve spatially precise measurements of endogenous DA release (Jaquins-Gerstl and Michael, 2015).

The development of a genetically encoded fluorescent sensors, which we call GPCR Activation-based (GRAB) sensors, has overcome the limitations of past methods and has enabled direct, rapid, sensitive, and cell-type specific detection of extracellular neurotransmitters. GRAB neurotransmitter sensors were initially developed by Dr. Yulong Li’s Lab at Peking University.

The sensors were engineered by coupling a conformationally sensitive circular-permutated EGFP (cpEGFP) to a selected human neurotransmitter receptor, such as DA receptor. For instance, GRAB_DA sensors were constructed by replacing the third intracellular loop (IL3) of the human Dopamine D1 (DRD1), D2 (DRD2) and D4 receptors (DRD4) with a cpGFP module from GCaMP6 (Patriarchi et al., 2018). Upon binding of neurotransmitters, such as dopamine, to the engineered receptor, a ligand-stabilized conformational change occurs in the receptor, which in turn results in an optical response of the conformation-sensitive fluorescent protein inserted in the receptor’s third intracellular loop (Sun et al., 2020). Therefore, changes in the fluorescence intensity of fluorescent proteins can be used to indicate dynamic changes in the concentration of neurotransmitters. These sensors enable real-time detection of endogenous neurotransmitters in acute brain slices of mice and in the intact brains of versatile animal models including flies, fish, and mice.


Figure 1. Schematic diagram showing the working principle of GRAB_DA Sensor. Upon binding of dopamine to the engineered receptor, a ligand-stabilized conformational change occurs in the receptor, which in turn results in an optical response of the conformation-sensitive fluorescent protein inserted in the receptor’s third intracellular loop.
 

• 1) High sensitivity;
• 2) High specificity;
• 3) High temporal and spatial resolution;
• 4) High signal-to-noise ratio;
• 5) Non-invasive;
• 6) No need to build additional detection equipment, it is consistent with the calcium signal detection system.

All GRAB sensors (including the latest ACh, DA, NE, 5-HT, Ado, ATP, VIP, CCK and eCB sensors) with AAV are available. To view the probe list, please click here or contact [email protected]. Please email [email protected] if the AAV you are looking for is not listed. We can design and package the AAV vectors to serve your special research needs.