Calcium indicator loading of neurons using single-cell electroporation

Area of Science:

• Neuroscience
• Cell Biology
• Biophysics

Background:

• Subcellular calcium (Ca2+) signaling is crucial for neuronal function.
• Accurate labeling of neurons with Ca2+ indicators is essential for studying these dynamics.
• Existing methods may have limitations in speed, efficiency, or specificity.

Purpose of the Study:

• To evaluate single-cell electroporation as a method for Ca2+ indicator loading in neurons.
• To assess the impact of electroporation on neuronal viability and function.
• To demonstrate the applicability of this technique in both in vitro and in vivo preparations.

Main Methods:

• Single-cell electroporation using brief voltage pulses delivered via glass pipettes.
• Loading of Ca2+ indicator dyes into individual neurons and small neuronal networks in rat neocortex.
• Simultaneous whole-cell recordings to assess neuronal viability and electrical properties post-electroporation.
• Functional assessment of Ca2+ transients in various neuronal compartments.

Main Results:

• Reliable and rapid (seconds) loading of somata and complete labeling of dendritic and axonal arborizations.
• High cell viability (approximately 85%) with rapid recovery from membrane permeabilization (within one minute).
• Electroporation did not alter the electrical properties of neurons.
• Normal Ca2+ transients were observed in dendrites, spines, and axonal boutons of electroporated cells.
• Successful application in vivo for targeted single-cell loading.

Conclusions:

• Electroporation is a simple, rapid, and effective method for Ca2+ indicator loading in individual neurons and small networks.
• The technique ensures high cell viability and preserves neuronal function, making it suitable for detailed Ca2+ dynamics studies.
• This approach is advantageous for functional imaging of subcellular Ca2+ signaling with minimal background staining.