Meeting Abstract
Identifying neuronal types within brains is key to understanding their roles in circuits underlying behavior. The brains of gastropod molluscs, such as the nudibranch Berghia stephanieae, contain a modest number of neurons (~7,000), many potentially identifiable by anatomical and neurochemical phenotypes. These traits make classifying every neuronal type feasible. Despite this, only a handful of neurons in gastropods have been identified and named. Gastropod brain development is determinate, but the precise soma locations and branching patterns are stochastic. This makes comparing brains and neurons across individual animals challenging. To address this obstacle, we are creating a standardized reference brain atlas for Berghia. We used CLARITY to clear whole animals and imaged them with lightsheet microscopy, leaving the brain in its true anatomical position. Clearing with CLARITY took ~2 weeks with minimal hands-on time. A lightsheet microscope easily accommodates the small slugs, allowing entire animal to be imaged in only a few minutes. We are currently aligning and averaging multiple 3D autofluorescent brain images together to create the reference brain. The CLARITY protocol preserves proteins in place, allowing us to use immunohistochemistry (IHC) sequentially on the same sample to start identifying neuronal types. So far we have labeled serotonin, small cardioactive peptide, and FMRFamide. Many neurons expressed only one of the 3 neurotransmitters we labeled with IHC. We found cells that are likely homologous to previously identified neurons in other nudibranchs based on position and labelling. Once the reference brain is completed, we will continue to add IHC and in situ hybridization labeling for other markers to identify all neuronal types in the Berghia brain. This protocol is likely applicable to other smaller invertebrates to create reference brain atlases.
