- • We developed an automated system that detects neurons belonging to specific populations in vitro or in situ, maps their physical locations in three-dimensional tissue specimens and then laser ablates the cell ‘targets’ one at a time, in sequence, while monitoring neural population activity electrophysiologically.
- • Two-photon Ca2+ imaging and image processing routines detect and validate target neurons based on rhythmic Ca2+ fluorescence activity patterns.
- • Visible-wavelength confocal imaging and image processing routines detect and validate target neurons that express genetically encoded fluorescent proteins.
- • High-intensity two-photon spot scanning vaporizes target neurons with specificity while minimizing damage to neighbouring tissue.
- • Physiological monitoring of network function is performed before, during and after the cell-specific laser ablations to measure the effects on network functionality in real time.
Abstract A key feature of neurodegenerative disease is the pathological loss of neurons that participate in generating behaviour. To investigate network properties of neural circuits and provide a complementary tool to study neurodegeneration in vitro or in situ, we developed an automated cell-specific laser detection and ablation system. The instrument consists of a two-photon and visible-wavelength confocal imaging setup, controlled by executive software, that identifies neurons in preparations based on genetically encoded fluorescent proteins or Ca2+ imaging, and then sequentially ablates cell targets while monitoring network function concurrently. Pathological changes in network function can be directly attributed to ablated cells, which are logged in real time. Here, we investigated brainstem respiratory circuits to demonstrate single-cell precision in ablation during physiological network activity, but the technique could be applied to interrogate network properties in neural systems that retain network functionality in reduced preparations in vitro or in situ.