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Related Experiment Video

Updated: Jun 5, 2025

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Generate and Analyze Three-Dimensional Dendritic Spine Morphology Datasets With SpineTool Software.

Anita Ustinova1, Ekaterina Volkova1, Anastasiya Rakovskaya1,2

  • 1Laboratory of Biomedical Imaging and Data Analysis, Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia.

Current Protocols
|December 6, 2024
PubMed
Summary

This study presents a protocol for analyzing dendritic spine morphology in mouse hippocampal neurons using 3D confocal microscopy and the SpineTool software. It details image processing, segmentation, and classification for understanding synaptic changes in health and Alzheimer's disease models.

Keywords:
classificationclusteringdendritic spinessegmentationsoftware

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Biophysics

Background:

  • Dendritic spine morphology reflects neuronal state and synaptic plasticity.
  • Alterations in dendritic spines are linked to neurodegenerative and neurodevelopmental disorders.
  • Accurate spine analysis is crucial for studying synaptic function and testing therapeutic strategies.

Purpose of the Study:

  • To outline a detailed protocol for imaging and analyzing dendritic spine morphology in mouse hippocampal neurons.
  • To establish an in vitro model of Alzheimer's disease using low amyloid toxicity.
  • To introduce and demonstrate the utility of SpineTool for 3D spine segmentation, classification, and analysis.

Main Methods:

  • Isolation, culturing, and fluorescent labeling of primary mouse hippocampal neurons.
  • Three-dimensional (3D) confocal microscopy for high-resolution imaging.
  • Image deconvolution to enhance resolution and reduce noise.
  • Utilizing SpineTool (open-source Python script) for 3D segmentation, feature extraction, and classification (manual, machine learning, k-means, DBSCAN).

Main Results:

  • Successful acquisition of 3D confocal images of dendritic spines from cultured neurons and brain slices.
  • Demonstrated effectiveness of image deconvolution in improving spine reconstruction.
  • SpineTool enables robust 3D segmentation and quantitative analysis of spine morphology (length, volume, surface area).
  • SpineTool facilitates classification and clustering of dendritic spines based on morphology.

Conclusions:

  • The protocol provides a comprehensive method for analyzing dendritic spine morphology in various experimental conditions.
  • SpineTool is a valuable open-source tool for quantitative analysis and classification of dendritic spines.
  • This methodology aids in understanding synaptic plasticity and pathological changes in neurological disorders like Alzheimer's disease.