Tracking connectivity maps in human stem cell-derived neuronal networks by holographic optogenetics

Area of Science:

• Neuroscience
• Stem Cell Biology
• Systems Biology

Background:

• Human induced pluripotent stem cells (hiPSCs) are crucial for modeling neuronal circuits and diseases.
• In vitro neuronal networks require extended culturing for functional maturation, limiting current research to immature networks.
• Assessing long-term functional features of hiPSC-derived neuronal networks is essential.

Purpose of the Study:

• To develop an improved glia-neuron co-culture protocol for long-term functional assessment of hiPSC-derived neuronal networks.
• To investigate the maturation of neuronal network activity using advanced stimulation techniques.
• To reveal functional neuronal connectivity motifs and their development over time.

Main Methods:

• Established an improved glia-neuron co-culture protocol within multi-electrode arrays for weekly electrical assessments.
• Utilized full-field optogenetic stimulation to analyze neuronal firing and burst activity.
• Employed single-cell holographic stimulation to map precise functional neuronal connectivity and activity propagation.

Main Results:

• Full-field optogenetic stimulation revealed an earlier onset of neuronal firing and burst activity compared to spontaneous activity.
• Stimulation enhanced the number of active neurons and their firing rates.
• Holographic stimulation enabled tracing of evoked activities from 400 individually stimulated neurons, revealing precise functional connectivity motifs that increased in number and strength with culture age.

Conclusions:

• The improved co-culture protocol and holographic stimulation setup provide a robust platform for analyzing hiPSC-derived neuronal networks.
• This method allows for in-depth investigation of network maturation and functional connectivity over extended periods.
• The findings offer a powerful tool for neurological disease modeling and drug screening using human neuronal networks.