The mid-brain raphe nucleus is a highly conserved structure present throughout vertebrates and holds a widespread interest because it is the source of the serotonergic network of the entire brain. Although variation exists among serotonergic neuronal population, still little is known about their differences. Even less is known about the non-serotonergic neuronal population. The precise projection of each population of neurons is also not identified. Studies in various animals have implicated raphe nucleus in functions including; sleep, arousal, fear, reward, aggression, and pain. Also, serotonin imbalance has been found to increase susceptibility to neurological disorders such as autism. However, which population of neurons is responsible for modulating any of these behaviors, and what are their roles is yet to identify. Therefore in this application, I will address some of these issues using six day old larval zebrafish. A much simpler but conserved brain structure to mammals, transparency in the earlier stages, huge clutch size and amenability to genetic modification make them an ideal system for the proposed research. Using the Burgess laboratory collection of transgenic and enhancer trap lines, I will identify various serotonergic and non-serotonergic population of neurons in the raphe nucleus in aim 1 by imaging. I will further characterize them with RNA-seq experiments followed by the in situ hybridization and generate their molecular profile. My preliminary data already suggest that there is multiple sub-populations of non- serotonergic neurons exist.
In aim 2, I will generate the projectome profile by stochastically labeling individual neurons in each of these sub populations using Gal4/Cre intersectional approach. To identify functional subregions, genetically encoded calcium indicator (GCaMP) will be expressed in the lines used in aim 1 and will be imaged while subjecting the larvae to flow stimulus. Neurons in the same functional subregions will show similar Ca++ activity. I will test the hypothesis that mutation in an autism candidate gene, solute carrier family 6, member 4 (slc6a1), a ?-amino acid butyric acid (GABA) transporter, causes persistent GABA neurotransmission resulting in serotonin imbalance and manifests autism like behavior in aim 3. First, I will generate mutants in the lines characterized in aim 1 and 2. I will check developmental defects by imaging population of the raphe neurons in mutants and comparing them to their wild type siblings. The functional defects will be tested similar to aim 2 because heightened sensory responsiveness is a common autism symptom. By the end of this study, I aim to generate clear functional subdivision of raphe neurons and identify their role in the autism that may be applicable for the therapeutic intervention.
The mid-brain raphe nucleus is the source of serotonergic network in vertebrate brains, however little is known about the neuronal subtypes, their projection pattern and their role in diseases that involve serotonin dysregulation such as autism. This project focuses on defining the subregions of raphe by categorizing the neuronal population and establishing their projection pattern. Furthermore, role of raphe neurons in manifestation of autism symptoms will also be established. The outcome of this research will be applicable to the other model systems and will also help understanding the neurotransmitter basis of autism.
