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Minimal neuronal circuit for action command generation and motor behaviour in Caenorhabditis elegans

Luka Železnik (2018) Minimal neuronal circuit for action command generation and motor behaviour in Caenorhabditis elegans. MSc thesis.

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    To understand how the nervous system functions, we have to understand its components and how those components interact with each other. Linking neural circuit motifs and structures to specific computations may potentially aid our understanding of how neural processing is achieved. C. elegans provides an excellent model system to understand micro-circuits; with its accessible genome and its small size, it is ideal addressing these questions. One of the important questions in this field is, at what level of organizational complexity do computations first arise? A related question is: what are the computational units that perform specific functions? To answer these questions, I built an experimental method wherein we could suppress – or silence – the whole nervous system and then reconstitute and record activities of individual neurons or small groups of neurons. For the silencing, I used genetically-expressed histamine-gated chloride channels (“HisCl”) which inhibit neurons by chloride ion influx, resulting in neuron hyperpolarization. A transgenic Cre-Lox strategy was used to make the expression specific by inverting the gene cassette to selectively prevent expression. To validate this approach, I first attempted to silence the whole nervous system, followed by whole nervous system reconstitution. If this technique achieves sufficient silencing and reconstitution, then the first experiment should fully paralyze animals, while the latter should completely reverse the paralysis. I fully paralyzed the worms by silencing their nervous system. This result suggests that the expression of the silencing transgene was indeed pan-neuronal, and imaging of a co-expressed fluorophore confirmed that the HisCl expression was taking place. However, the imaging revealed that the gene cassette inversion did not occur in all cells and was incomplete, suggesting that at the current state the efficiency of the gene inversion needed to be improved. Thus it was not surprising that the reconstitution of the nervous system only partially reversed the paralysis. This was expected since we were testing animals not yet optimized for expression efficiency but was a necessary first step. The project revealed several flaws in our approach, both in the experimental setup and the quantification methods. The main improvement suggested by high mosaicism (i.e., incomplete expression in all neurons) and variability between the transgenic lines is the need to integrate the transgene into the genome, which is known to reduce these problems. The results also suggest a strategy other than Cre-Lox might be useful. Further work is required to establish a method that will allow us to successfully perform the experiments that we envisioned during the conception of the project.

    Item Type: Thesis (MSc thesis)
    Keywords: C. elegans, neuronal dynamics, Cre-Lox, histamine-gated chloride channel
    Number of Pages: 52
    Language of Content: Slovenian
    Mentor / Comentors:
    Mentor / ComentorsIDFunction
    prof. dr. Manuel ZimmerMentor
    Link to COBISS: https://plus.si.cobiss.net/opac7/bib/12330569
    Institution: University of Ljubljana
    Department: Faculty of Education
    Item ID: 5611
    Date Deposited: 28 Feb 2019 08:38
    Last Modified: 28 Feb 2019 08:38
    URI: http://pefprints.pef.uni-lj.si/id/eprint/5611

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