On-Demand In Vivo Phototagging for Calcium-Guided Neuron Labeling
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Solution Overview
Problem
Current methods are limited in their ability to precisely tag single functionally identified cortical glutamatergic pyramidal neurons (PNs) in vivo, due to spatial resolution issues, deficiencies in targeting neurons with high baseline intracellular Ca2+ levels, and the inability to label neurons that decrease activity in response to behavioral states or sensory stimuli, hindering the understanding of gene expression's role in circuit function and behavior.
Innovation Solution
A nucleic acid molecule comprising a transcription regulatory element linked to an open reading frame encoding GCaMP7f, a ribosomal skipping peptide, and a fusion protein of a nuclear protein and photoactivatable red fluorescent protein, delivered via an expression vector, such as an AAV, for simultaneous calcium measurement and labeling, combined with two-photon phototagging and RNA sequencing to analyze functional profiles in cortical circuits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If previous Ca2+ and light-dependent labeling methods are used, then transiently active neurons can be labeled, but spatial resolution is insufficient and neurons with high baseline intracellular Ca2+ levels cannot be targeted
Solution Approach 1:
The labeling process is segmented into distinct phases: first GCaMP7f expresses calcium activity in real-time, then a separate photoactivation step selectively tags only the calcium-active neurons with PAmCherry. This segmentation allows precise temporal and spatial control, resolving the contradiction between measurement precision and labeling accuracy by separating detection from permanent tagging.
Solution Approach 2:
GCaMP7f serves as an intermediary that temporarily reports calcium activity without permanently labeling the neuron. This intermediary enables indirect detection of neuronal activity states, allowing selective identification of target neurons based on their calcium dynamics before applying the permanent PAmCherry label, thus achieving both high spatial resolution and reliable targeting.
2Adaptability or versatility
If previous labeling methods are used, then some active neurons can be tagged, but the ability to label neurons that decrease activity in response to behavioral states or sensory stimuli is lost
Solution Approach 1:
The system dynamically adapts to different neuronal activity patterns by using GCaMP7f's real-time calcium reporting capability during the imaging phase. Neurons exhibiting any calcium transient pattern (including decreases) can be identified and subsequently tagged with PAmCherry, providing versatile labeling coverage while maintaining precise detection of various activity patterns through the dynamic GCaMP7f readout.
3Manufacturing precision
If photoactivatable fluorescent proteins are used for single-cell precision tagging, then spatial precision is improved, but the method has been deployed with limited success in densely packed neurons
Solution Approach 1:
The method performs preliminary calcium imaging with GCaMP7f to identify and characterize target neurons before applying the photoactivation step. This preliminary action allows optimization of photoactivation parameters for each specific neuronal target, ensuring high labeling precision while maximizing success rate by avoiding blind photoactivation in densely packed regions without prior identification.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise, in vivo labeling and functional profiling of densely packed glutamatergic neurons, allowing for molecular characterization of functionally distinct neurons in behaving animals, overcoming spatial and temporal resolution limitations of previous methods.
Implementation Method 1
GCaMP7f, a ribosomal skipping peptide, and a fusion protein of a nuclear protein and photoactivatable red fluorescent protein
Implementation Method 2
photoactivatable red fluorescent protein
Implementation Method 3
two-photon phototagging
Data Source
AI summary
Nucleic acid molecules comprising at least one transcription regulatory element operably linked to an open reading frame, wherein the open reading frame encodes a single RNA transcript encoding GCaMP7f, a ribosomal skipping peptide, and a fusion protein of a nuclear protein and photoactivatable red fluorescent protein are provided. Expression vectors and cells comprising the nucleic acid molecules are also provided, as are methods of using the nucleic acid molecules for simultaneous labeling and measuring calcium and analyzing a target cell.