Rhodopsins
are seven-transmembrane photoreceptor proteins that
bind to the retinal chromophore and have been utilized as a genetically
encoded voltage indicator (GEVI). So far, archaerhodopsin-3 (AR3)
has been successfully used as a GEVI, despite its low fluorescence
intensity. We performed comparative and quantitative fluorescence
analyses of 15 microbial rhodopsins to explore these highly fluorescent
molecules and to clarify their fluorescence mechanism. These rhodopsins
showed a wide range of fluorescence intensities in mouse hippocampal
neurons. Some of them, GR, HwBR, IaNaR, MR, and NpHR, showed fluorescence
intensities comparable with or higher than that of AR3, suggesting
their potential for GEVIs. The fluorescence intensity in neurons correlated
with that of the bright fluorescent photointermediate such as a Q-intermediate
(R = 0.75), suggesting that the fluorescence in neurons
originates from the fluorescence of the photointermediate. Our findings
provide a crucial step for producing next-generation rhodopsin-based
GEVIs.
Membrane potential is the critical parameter that reflects the excitability of a neuron, and it is usually measured by electrophysiological recordings with electrodes. However, this is an invasive approach that is constrained by the problems of lacking spatial resolution and genetic specificity. Recently, the development of a variety of fluorescent probes has made it possible to measure the activity of individual cells with high spatiotemporal resolution. The adaptation of this technique to image electrical activity in neurons has become an informative method to study neural circuits. Genetically encoded voltage indicators (GEVIs) can be used with superior performance to accurately target specific genetic populations and reveal neuronal dynamics on a millisecond scale. Microbial rhodopsins are commonly used as optogenetic actuators to manipulate neuronal activities and to explore the circuit mechanisms of brain function, but they also can be used as fluorescent voltage indicators. In this review, we summarize recent advances in the design and the application of rhodopsin-based GEVIs.
Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active-zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between Src homology three and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of SV organization and spontaneous release.
Synaptic vesicles (SVs) are clustered in the presynaptic terminals and consistently trafficking along axons. Based on their release features, SVs are classified into different "pools". Imaging of SVs that are traveling among multiple presynaptic terminals has helped define a new pool named "SV superpool". Here we describe a Fluorescent Recovery After Photobleaching (FRAP) approach to elucidate the relationship between SVs from the super-pool with SV clusters at presynaptic terminals. This method is powerful to investigate SV mobility regulation mechanisms.
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