Fluorescent Biosensors for Neurotransmission and Neuromodulation

Neurotransmitters and neuromodulators

Neurotransmitters and neuromodulators are chemical messengers that play essential roles in signal transmission within neural circuits. Neurotransmitters are released from the axon terminals of presynaptic neurons and rapidly excite (e.g., glutamate) or inhibit (e.g., γ-aminobutyric acid, GABA) postsynaptic neurons on a sub-second timescale. They are stored in synaptic vesicles and released into the synaptic cleft in response to action potentials. In contrast, neuromodulators are diffusible signaling molecules that regulate the activity of populations of neurons and operate over both fast and slow timescales. Importantly, even classical fast neurotransmitters such as glutamate do not always function exclusively through point-to-point synaptic transmission. Neurotransmitters can diffuse from the synaptic cleft into the extracellular space and activate extrasynaptic receptors at considerable distances, a process known as volumetric transmission.

Neurotransmitters and neuromodulators are fundamental to cognition and behavior. The relationship between chemical signaling in the brain and complex animal behavior can now be investigated using modern fluorescent biosensors, which provide high spatiotemporal resolution for visualizing rapid neurotransmitter dynamics within neural circuits of behaving animals.

Fluorescent proteins

Fluorescent proteins (FPs) are fundamental components of modern biosensors. Two principal strategies are used to incorporate FPs into biosensor designs. The first relies on Förster resonance energy transfer (FRET) between two FPs. In FRET-based sensors, excitation of a donor FP leads to non-radiative transfer of energy to a nearby acceptor chromophore, producing a measurable fluorescence change. The second strategy uses circularly permuted fluorescent proteins (cpFPs) , in which the original protein sequence is rearranged while preserving the overall fold. Specific regions of cpFPs tolerate insertion of sensing domains, and conformational changes in these inserts can strongly modulate fluorescence intensity. Circular permutation also alters the orientation of the chromophore relative to fusion partners, a property that can be exploited to optimize FRET-based biosensors by incorporating cpFPs. Fluorescent biosensors can be delivered to the brain using viral vectors, such as AAV and monitored in behaving animals with a range of optical techniques, including fiber photometry and multichannel fiber photometry, stationary two-photon (2P) microscopy, and miniaturized head-mounted microscopes. Neurotransmitter dynamics in response to visual, auditory, or olfactory stimuli can be imaged in restrained animals, while wireless head-mounted microscopes enable biosensor imaging in the brains of freely moving animals.

Recently developed fluorescent biosensors for glutamate, dopamine, acetylcholine, adrenaline and GABA allow to detect neuronal activity in vivo with high spatiotemporal precision. Single-FP-based intensiometric biosensors represent the most useful group of the biosensors because they are monochromic and have the higher dynamic range than FRET-based, thus, enabling spectral multiplexing with other biosensors or optogenetic tools and imaging of neuronal activity in vivo, respectively.

Biosensors for neurotransmitters and neuromodulators (Anna et al, 2019PMID: 31708747)

FRET-based biosensors	FRET pair	Relative change of FRET ratio,ΔR/R (%)	K_d (μM)	Conditions used to measure ΔR/R and K_d	Substance (neurotransmitter or neuromodulator)	Template for sensing domain	In vivo use	References
SuperGluSnFR	ECFP-Citrine	44	2.5	Cultured neurons, 1P microscopy	Glutamate	GltI	Not tested	Hires et al., 2008
M1-cam5	ECFP-EYFP	10	Not determined	HEK293 cells, 1P microscopy	Acetylcholine	M1mAChR		Markovic et al., 2012
GlyFS	EGFP-Venus	20	20	Brain slices, 2P microscopy	Glycine	Atu2422 (AYW mutant)		Zhang et al., 2018

Single-FP-based biosensors	Circularly permuted FP	Relative change of fluorescence,ΔF/F (%)	K_d (μM)	Conditions used to measure ΔF/F and K_d	Substance (neurotransmitter or neuromodulator)	Template for sensing domain	In vivo use	References
iGluSnFR	cpGFP	103	4.9	Cultured neurons, 1P microscopy	Glutamate	GltI	Imaging of dendritic spines	Marvin et al., 2013
SF-iGluSnFR A184V	sfGFP	69	0.6					Marvin et al., 2018
SF-iGluSnFR S72A		250	34
SF-Azurite-iGluSnFR	Azurite	66	46
SF-Venus-iGluSnFR	Venus	66	2
SF-mTurquoise2-iGluSnFR	mTurquoise	90	41
iGABASnFR	sfGFP	250	9	Purified protein, fluorimeter	GABA	Pf622	Imaging of single neurons	Marvin et al., 2019
iGlu_f	EGFP	100	137	HEK293 cells, stopped-flow	Glutamate	GltI	Not tested	Helassa et al., 2018
iGlu_u		170	600
R-iGluSnFR1	mApple	−33	11	Purified protein, fluorimeter				Wu et al., 2018
R-ncp-iGluSnFR1			0.9
GACh	EGFP	90	2	HEK293 cells, 1P microscopy	Acetylcholine	M₃R	Imaging of single neurons	Jing et al., 2018
GRAB_NE1m		230	1.9		Norepinephrine	α2AR	Aggregated fluorescence signal	Feng et al., 2019
GRAB_NE1h		150	0.093
Nb80-GFP		Not determined	Not determined	Not applicable		β2AR/Nb80	Not tested	Irannejad et al., 2013
OR-sensor	EGFP	Not determined	Not determined	Not applicable	Activation of μ and δ ORs	μ and δ ORs/Nb33	Not tested	Stoeber et al., 2018
iATPSnFR	spGFP	150	630	Cultured neurons, 1P microscopy	ATP	ε subunit of FOF1 ATPase from Bacillus PS3	Imaging of single astrocytes	Lobas et al., 2018
dLight1.1	EGFP	230	0.33	HEK293 cells, 1P microscopy	Dopamine	DRD1 (inserted into the ICL3)	Aggregated fluorescence signal	Patriarchi et al., 2018
dLight1.2		340	0.77
DA1m		90	0.13			DRD2 (inserted into the ICL3)		Sun et al., 2018
DA1h			0.01

AAV Biosensors

AAV biosensors are genetically encoded reporter systems delivered by AAV vectors that convert intracellular biological events into measurable optical or luminescent signals in living cells, tissues, or animals. They allow non-invasive, cell-type–specific, and longitudinal monitoring of signaling pathways, neuronal activity, and cellular states in vivo—which is why they are becoming a core tool in neuroscience, immunology, and gene-therapy development.

In practice, an AAV biosensor is built from four components:
a promoter (to define which cells are measured), a sensor module (that detects a biochemical activity), a reporter (fluorescent or luminescent), and an AAV capsid (that determines which tissues are transduced). AAV makes biosensors usable in vivo, not just in cultured cells:

Stable, long-term expression (months to years)
Cell-type specificity using promoters (Syn, GFAP, CamKII, CD4, etc.)
Deep tissue access using engineered capsids (BBB, retina, muscle, lung)
Repeated measurements in the same animal