A leech brain in the dish: a method for detailed analysis of specifically labeled single cells

摘要

One of the major goals in neuroscience is to identify the enormous diversity of neurons, depict their heterogeneity, andexplore their function under normal and pathological conditions. To date, the repetitive study of specific neurons in thevertebrate nervous system is almost impossible. Invertebrate nervous systems, on the other hand, have provided anopportunity to study single identified neurons for decades. However, simple methods to study the identified neurons incell culture are still lacking.In this research study we developed a simple method, based on microinjection, to study and manipulate single identifiedneurons in culture. The concept behind this method is to utilize the advantages and simplicity of the leech nervoussystem. Within the leech ganglion, neurons are arranged in a characteristic and stereotypical manner. Their location, size,and biophysical properties are highly characterized. This allows us to identify and label specific cells of interest prior toplating. These cells can be later identified throughout their culture development solely by fluorescent imaging thousandsof cells in a cell culture consisting of neuronal and non-neuronal cell populations. Here, we present a proof of concept forthis method. We demonstrate neuronal viably by following the procedure up to 7 days and by introducing differentmolecules to different neurons of interest. This method can easily be adapted to other invertebrate brains and opens upnew possibilities for precise manipulation, study, and characterization of specific individual neurons in cell culture.