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

One of the major goals in neuroscience is to identify the enormous diversity of neurons, depict their heterogeneity, and explore their function under normal and pathological conditions. To date, the repetitive study of specific neurons in the vertebrate nervous system is almost impossible. Invertebrate nervous systems, on the other hand, have provided an opportunity to study single identified neurons for decades. However, simple methods to study the identified neurons in cell culture are still lacking. In this research study we developed a simple method, based on microinjection, to study and manipulate single identified neurons in culture. The concept behind this method is to utilize the advantages and simplicity of the leech nervous system. 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 to plating. These cells can be later identified throughout their culture development solely by fluorescent imaging thousands of cells in a cell culture consisting of neuronal and non-neuronal cell populations. Here, we present a proof of concept for this method. We demonstrate neuronal viably by following the procedure up to 7 days and by introducing different molecules to different neurons of interest. This method can easily be adapted to other invertebrate brains and opens up new possibilities for precise manipulation, study, and characterization of specific individual neurons in cell culture.