Automated cell‐specific laser detection and ablation of neural circuits in neonatal brain tissue

Abstract

Key points• 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 Ca 2+ imaging and image processing routines detect and validate target neurons based on rhythmic Ca 2+ 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 Ca 2+ 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.

Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice

“…Subsequent detection of overlaying ROIs at the superficial focal plane (−10 µm), which also pass the circularity test, are nonetheless rejected by the priority rule and thus colored blue ( D 2 , circled ROIs). These criteria for target detection are more fully described in ‘Materials and methods’ and Wang et al (2013). DOI: http://dx.doi.org/10.7554/eLife.03427.005

10.7554/eLife.03427.006Figure 1—figure supplement 3.Average number of Dbx1 neurons detected at each acquisition depth from z = 0 (surface) to z = −80 µm in preBötC-surface slices and control slices with the ventral respiratory column (VRC) exposed at the slice surface.The number of Dbx1 neurons detected per focal plane per side (in 10-µm increments of the focal plane) is shown individually for each individual experiment (graygray unfilled circles) along with the mean ±SD for all experiments (black unfilled squares with black lines showing SD). DOI: http://dx.doi.org/10.7554/eLife.03427.006

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“…Potential targets were evaluated on the basis of shape to differentiate cell bodies from auto-fluorescent debris, and to reject the fluorescence from dendrites and neuropil whose somata were detectable in adjacent focal planes (Wang et al, 2013) (Figure 1—figure supplement 2). The map of ROIs for validated cell targets was retained at each focal plane (Figure 1C—figure supplements 1B and 2, red ROIs).…”

“…The ablation is confirmed if fluorescence is detected in the band 560–615 nm, which reflects presumed water vapor in the cell cavity and excludes infrared reflections of the long-wavelength laser (Figure 2—figure supplement 1A) (Wang et al, 2013). In addition, lesioned targets disappear from the fluorescence image (Figure 2—figure supplement 1B), and their pre-lesion bright field image (Figure 2—figure supplement 1C) is replaced post-lesion by a pock mark (Figure 2—figure supplement 1D).…”

Facing the challenge of mammalian neural microcircuits: taking a few breaths may help

“…One novel approach for preBötC microcircuit dissection is sequential targeted removal of neurons by laser photoablation (Hayes et al 2012). Sequential ablation of ß120 inspiratory-modulated neurons in vitro results in the loss of rhythmic motor output (Hayes et al 2012;Wang et al 2013). When lesioning is restricted to Dbx1 + (glutamatergic) preBötC neurons, rhythm stops after ablating ß85 neurons, suggesting that rhythmogenic circuits are more sensitive to the specific loss of Dbx1 + neurons than to generic inspiratory-modulated neurons , half of which are inhibitory (Winter et al 2009).…”

“…When lesioning is restricted to Dbx1 + (glutamatergic) preBötC neurons, rhythm stops after ablating ∼85 neurons, suggesting that rhythmogenic circuits are more sensitive to the specific loss of Dbx1 + neurons than to generic inspiratory‐modulated neurons (Wang et al . , ), half of which are inhibitory (Winter et al . ).…”