研究目的
To characterize the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina and utilize optic nerve transection (ONT) as a model to assess changes in intracellular calcium in retinal ganglion cells (RGCs) after axonal injury.
研究成果
GCaMP3 is specifically expressed in RGCs and remains so after ONT. Functional responses to KA are reduced prior to structural loss of RGCs, indicating that anatomical markers may underestimate dysfunction. This suggests GCaMP3 is useful for studying RGC function in disease models.
研究不足
The study may have limitations in generalizing to other injury models or chronic conditions, and the specificity of GCaMP3 expression could be affected by cellular changes post-injury. The use of transgenic mice may not fully represent human retinal physiology.
1：Experimental Design and Method Selection:
The study used immunohistochemistry to determine the specificity of GCaMP3 expression in retinal ganglion cells (RGCs) and calcium imaging to characterize functional responses to kainic acid (KA) in control and optic nerve transection (ONT) groups.
2：Sample Selection and Data Sources:
Thy1-GCaMP3 transgenic mice (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) were used, with ONT performed in a subset 3, 5, or 7 days before sacrifice. Retinas were isolated and processed.
3：List of Experimental Equipment and Materials:
Equipment includes microscopes (e.g., Axio Imager.M2, Axioskop), cameras (e.g., Axiocam 506, Sensicam PCO), light sources (e.g., X-Cite 120Q, Lambda-LS), antibodies (e.g., anti-GFP, anti-RBPMS), and chemicals (e.g., kainic acid, Hibernate A solution).
4：Experimental Procedures and Operational Workflow:
Retinas were fixed and stained for immunohistochemistry. For calcium imaging, retinas were mounted and superfused with KA solutions while recording fluorescence changes. ONT was performed under anesthesia.
5：Data Analysis Methods:
Cell densities and colocalization were quantified using software (e.g., Zen2Lite). Calcium transient amplitudes (ΔF/F0) were calculated, and statistical analysis was performed using ANOVA and Tukey's test with software like Prism6.
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Axio Imager.M2
Axio Imager.M2
Carl Zeiss
Microscope for obtaining tiled fluorescence images
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Axiocam 506 camera
Axiocam 506
Carl Zeiss
Digital camera for capturing fluorescence images
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X-Cite 120Q
X-Cite 120Q
Excelitas Technologies
Fluorescence excitation light source
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Axioskop
Axioskop
Carl Zeiss
Upright microscope for calcium imaging
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Sensicam PCO
Sensicam PCO
PCO
Charge-coupled device (CCD) camera for capturing fluorescence
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Hibernate A
Hibernate A
ThermoFisher Scientific
Solution for maintaining retinas
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Anti-GFP antibody
A21311
ThermoFisher
Antibody for immunohistochemistry to enhance GCaMP3 fluorescence
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Anti-GABA antibody
A2052
Sigma-Aldrich Corp.
Antibody for GABAergic amacrine cell marker
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Zen2Lite
Zen2Lite
Carl Zeiss
Software for quantifying cell counts and colocalization
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Lambda-LS
Lambda-LS
Sutter Instruments
Xenon lamp for exciting GCaMP3
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Kainic acid
R&D Systems
Chemical used to evoke calcium transients
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Anti-RBPMS antibody
1832-RBPMS
PhosphoSolutions
Antibody for RGC-specific marker in immunohistochemistry
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Anti-ChAT antibody
AB144P or 315-ChAT
Millipore or PhosphoSolutions
Antibody for cholinergic amacrine cell marker
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Anti-syntaxin antibody
1990-STX
PhosphoSolutions
Antibody for amacrine cell marker
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Anti-GAD67 antibody
MAB5406
Millipore
Antibody for GABAergic amacrine cell marker
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Secondary antibodies
e.g., Cy3, AlexaFluor 633, AlexaFluor 647
Jackson ImmunoResearch or ThermoFisher
Secondary antibodies for immunohistochemistry
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VectaShield
VectaShield
Vector Laboratories
Antifade media for mounting retinas
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Axon Imaging Workbench 4
Axon Imaging Workbench 4
Molecular Devices
Software for recording and analyzing calcium imaging data
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ClampFit 10.4
ClampFit 10.4
Molecular Devices
Software for analyzing calcium transient amplitudes
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Prism6
Prism6
GraphPad
Software for statistical analysis
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