Genetically encoded fluorescent sensors for studying healthy and diseased nervous systems

Abstract

Neurons and glia are functionally organized into circuits and higher-order structures via synaptic connectivity, well-orchestrated molecular signaling, and activity-dependent refinement. Such organization allows the precise information processing required for complex behaviors. Disruption of nervous systems by genetic deficiency or events such as trauma or environmental exposure may produce a diseased state in which certain aspects of inter-neuron signaling are impaired. Optical imaging techniques allow the direct visualization of individual neurons in a circuit environment. Imaging probes specific for given biomolecules may help elucidate their contribution to proper circuit function. Genetically encoded sensors can visualize trafficking of particular molecules in defined neuronal populations, non-invasively in intact brain or reduced preparations. Sensor analysis in healthy and diseased brains may reveal important differences and shed light on the development and progression of nervous system disorders. We review the field of genetically encoded sensors for molecules and cellular events, and their potential applicability to the study of nervous system disease.

Figure 1. Probing molecular and cellular events in neurons

Action potentials are propagated by voltage-gated ion channels. At synapses, calcium influx during membrane depolarization can trigger neurotransmitter release via synaptic vesicle trafficking. Neurotransmitters, such as glutamate, then bind to post-synaptic ligand-gated ion channels, directly gating ion current, or to metabotropic receptors, activating ion channels through a G-protein coupled second messenger cascade. The action of second messengers, such as cyclic AMP (cAMP) and Ca²⁺, regulates downstream pathways, primarily through kinase signaling.

Figure 2. Schematic of genetically encoded fluorescence sensors

a) A biosensor consists of a recognition element and a reporter element. Upon target binding, the conformational change of the recognition element is transduced into an optical readout by the reporter element. b) Genetically encoded calcium indicators (GECIs). Representatives shown from the GCaMP and cameleon families [133]. Upon calcium binding, conformational change of the calmodulin/M13 complex results in modulation of fluorescence of circularly permuted GFP or of FRET between a CFP / YFP pair. c) The vesicle trafficking pH sensor, synapto-pHluorin [61]. The fluorescence of synapto-pHluorin is quenched by the acidic intraluminal environment of synaptic vesicles (pH ~ 5.6). During neurotransmitter release, vesicles fuse with the plasma membrane, exposing the lumen to the neutral pH of the extracellular environment (pH ~ 7.4), causing a dramatic increase in fluorescence intensity. The fluorescence intensity is then quenched once again after reacidification. d) The genetically encoded glutamate sensor, SuperGluSnFR[54]. The glutamate-binding protein GltI is sandwiched between a pair of FRET protein, ECFP and Citrine. e) Targeting of the fluorescent protein voltage sensor, VSFP2.1, to the plasma membrane [109]. The FRET pair ECFP and Citrine is attached to the C-terminus of the four-transmembrane voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP).

Figure 3. Imaging neurological disorders

a) Neurons labeled with genetically encoded sensors can be visualized by in vivo imaging. b) Schematic representation of sensor response to acute challenge in in vivo mouse preparation.