Robust automated protocol for extraction and comparison of single neuron morphology
Hiroyuki Ai (Fukuoka University), Stephan Haupt (University of Tokyo), Philipp Rautenberg (Ludwig-Maximilians-Universität München), Michael Stransky (Ludwig-Maximilians-Universität München), Thomas Wachtler (Ludwig-Maximilians-Universität München), Hidetoshi Ikeno (University of Hyogo)
LSM image stacks containing the entire morphology of single neurons are first subjected to a two-step segmentation. In the first step, brightness and contrast are adjusted to compensate for differences in noise and background levels among individual data sets, and binarization is applied. In the next step, extracted branching structures are traced based on the SSDT method using our software SIGEN (Yamazaki et al., 2008, doi: 10.1016/j.neucom.2005.12.042). SIGEN does not extract a wire model but also determines cylinder diameters for extracted segments. In this step, actual neuronal branch elements and false positive elements are still intermingled. Detected segments are then scrutinized and connected to the main branch based on two parameters, volume threshold (VT) and distance threshold (DT), finally resulting in cylinder models of the neurons. The final radius of cylinder elements corresponding to the thickness of a neurite segment is assigned by averaging the number of extracted pixels in a direction perpendicular to the skeleton center line within an element.
We applied our method to an identified interneuron in the honeybee auditory system. We compared the number of branches and estimated axial resistances of cylinder segments of neuron models extracted manually to results from our automated extraction protocol. We also investigated the effect of VT and DT on branch extraction success. The number of branches, especially in the fine dendritic areas, was clearly increased (up to 23%) by tuning of DT and VT. Our findings demonstrate how using well-defined parameters permits repeated and reproducible extraction of neuron morphologies and minimizes variability in reconstruction resulting from differences in the extraction process.
This research was supported by Strategic International Cooperative Program, Japan Science and Technology Agency (JST) and the German Federal Ministry of Education and Research (BMBF, grant 01GQ1116).