One of the goals of neuroscience is to classify all of the neurons in the brain. Neuronal types can be defined using a combination of morphology, electrophysiology, and gene expression profiles. Gene expression profiles allow differentiation between cells that share similar characteristics. Leveraging the advantage of Berghia stephanieae (Gastropoda; Nudibranchia), which has around 28,000 neurons, I constructed high-throughput single-neuron transcriptomes for its whole brain. I produced a single-cell dissociation protocol and a custom data analysis pipeline for data of this nature. Around 129,000 cells were collected from 18 rhinophore ganglia and 20 circumesophageal ring ganglia (brain), consisting of the cerebropleural, pedal, and buccal ganglia. Messenger RNA libraries were constructed using the 10X Genomics’ Chromium platform. After library preparation, around 1,000 cells were recovered and sequenced. The HTStream package was utilized to trim off unwanted sequences from the raw reads and remove PCR duplicates and other contamination, then the salmon alevin package was employed to construct gene-by-cell matrices containing all the transcripts for each gene in each cell. The Seurat pipeline was used to extract this expression data from the matrices, normalize it, and perform dimensionality reduction. The cells were clustered based on similarities in their gene expression profiles. The cells formed eight clusters on a UMAP graph, each having distinct marker genes. Additionally, one cluster was composed of almost exclusively cells from the rhinophore ganglia, accounting for 30% of all rhinophore ganglion cells in the sample. Cells from the rhinophore ganglia are as heteregenous as cells from the rest of the brain, with cells forming six clusters. Cell populations that express the same neurotransmitter were identified for a wide range of both small-molecule neurotransmitters and neuropeptides. In a separate project, the locomotion of Berghia was recorded over 9 days with 2 lighting regimes: LD first and DD first. The results suggest that locomotion of Berghia is governed by circadian clock and that Berghia is nocturnal. Hunger state likely plays a role in modulating this circadian rhythm.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-2077 |
| Date | 01 February 2021 |
| Creators | Bui, Thi |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
| Rights | http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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