Protocol for semi-automatic quantitative bioimaging analysis of synapse loss.

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• Synapse loss is a key feature of neurodegenerative diseases, including Alzheimer's disease, and is linked to cognitive decline.
• Quantifying synapse loss in experimental models is essential for understanding disease mechanisms and developing treatments.

Purpose of the Study:

• To develop and validate a semi-automated workflow for quantifying synapse loss in primary mouse neurons.
• To establish a protocol for analyzing synaptic changes following genetic manipulation, such as Bin1 knockdown.

Main Methods:

• Primary mouse neurons were cultured and treated with short hairpin RNA (shRNA)-expressing lentivirus.
• Immunofluorescence labeling was used to detect excitatory and inhibitory presynaptic markers.
• Image acquisition and analysis were performed using Fiji/ComDet macros for region of interest selection, neurite length measurement, and puncta detection.

Main Results:

• The workflow enables the quantification of synapse density, size, and intensity.
• Synaptic changes were successfully analyzed following Bin1 knockdown in primary mouse neurons.
• The protocol provides a robust method for assessing synaptic integrity.

Conclusions:

• The developed semi-automated workflow offers a reliable method for quantifying synapse loss in neuronal cultures.
• This protocol can be applied to study synaptic pathology in various neurodegenerative conditions.
• Accurate quantification of synaptic changes is vital for advancing research in Alzheimer's disease and related disorders.