研究目的
To develop a chemogenetic approach for optical monitoring of voltage in neurons using a semisynthetic tethered voltage indicator based on Nile Red, enabling genetic targeting and detection of supra- and subthreshold neuronal activity.
研究成果
Nile Red derivatives, when tethered to small protein tags like ACP-tag, exhibit significant voltage sensitivity and can be genetically targeted to neurons for optical monitoring of membrane potential. The probes enable detection of action potentials and subthreshold activity with fast kinetics and high fidelity, comparable to existing methods but with advantages in specificity and reduced light power requirements. Future work should investigate the mechanisms of voltage sensitivity and explore applications in subcellular targeting.
研究不足
The SNAP-tag targeted probes showed negligible voltage sensitivity due to potential interaction with hydrophobic surfaces of the tag rather than the membrane. The mechanism underlying Nile Red voltage sensitivity (e.g., solvatochromism, electrochromism) is not fully understood and requires future exploration. Application may be limited by the need for genetic modification of cells.
1：Experimental Design and Method Selection:
The study designed a hybrid voltage sensor combining a synthetic voltage indicator (Nile Red derivatives) with genetically encoded protein tags (SNAP-tag and ACP-tag) for targeted localization. Voltage sensitivity was assessed using whole-cell voltage clamp and epi-fluorescence imaging.
2：Sample Selection and Data Sources:
HEK293T cells and dissociated dorsal root ganglion (DRG) sensory neurons were used. Cells were transfected or infected with recombinant adeno-associated virus (AAV) for tag expression.
3：List of Experimental Equipment and Materials:
Equipment includes epi-fluorescent microscope, confocal microscope, whole-cell voltage clamp setup. Materials include Nile Red derivatives (e.g., NR12S, CoA-PEGn-NR), protein tags (SNAP-tag, ACP-tag), and reagents for synthesis and labeling.
4：Experimental Procedures and Operational Workflow:
Cells were labeled with probes (e.g., 500 nM NR12S for 7 minutes), subjected to voltage steps or current injections, and imaged at high frame rates (e.g., 333 fps). Fluorescence changes were recorded simultaneously with electrophysiological measurements.
5：Data Analysis Methods:
Data were analyzed for fractional fluorescence change (ΔF/F%), signal-to-noise ratio (SNR), kinetics (rise and decay times), and linearity (R2 values). Statistical analysis included mean ± standard deviation or confidence intervals, and One-Way ANOVA for comparisons.
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