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

master's thesis

Luka Železnik (Avtor), Manuel Zimmer (Mentor)

Povzetek

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.

Ključne besede

C. elegans;

Podatki

Jezik:	Angleški jezik
Leto izida:	2018
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UL PEF - Pedagoška fakulteta
Založnik:	[L. Železnik]
UDK:	612.822:575.16(043.2)
COBISS:	12330569
Št. ogledov:	1291
Št. prenosov:	222
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Slovenski jezik
Sekundarni naslov:	Minimalno nevronsko omrežje za ustvarjanje ukazov dejanj in motoričnega vedenja pri Caenorhabditis elegans
Sekundarni povzetek:	Da bi razumeli kako deluje živčni sistem, je potrebno najprej razumeti delovanje posameznih komponent in interakcij med temi komponentami. Mikro-omrežja so pomembna organizacijska enota živčnega sistema. Potencialno ugotoviti povezavo med vzorci znotraj teh mikro-omrežji in specifičnimi računskimi operacijami, bi bil pomemben napredek za nevroznanost. C. elegans je odličen model za raziskovanje na tovrstnem področju, zaradi genetskih orodji, ki so na voljo in majhnosti organizma. Eno izmed ključnih vprašanj na tem področju je, na katerem nivoju kompleksnosti se pojavijo prve računske operacije. Povezano vprašanje pa je katere organizacijske enote omrežja opravljajo katere računske naloge. Da bi odgovorili na ta vprašanja smo se lotili ustvarjanja eksperimentalnega orodja, ki bi nam omogočilo utišati celoten nevronski sistem in v nadaljevanju omogočilo delovanje zgolj posameznih delov. Da bi dosegli to utišanje nevronskega sistema smo uporabili transgensko izražene kloridne kanale, ki nevrone utišajo zaradi vdora kloridnih ionov in posledične hiperpolarizacije. Za samo selektivno delovanje posameznih delov, pa smo uporabili metodo Cre-Lox, ki omogoča selektivno inverzijo željenega transgena. Uspelo nam je doseči popolno paralizo, ki je posreden indikator polnega utišanja. Prav tako rezultati kažejo, da je gen uspešno izražen v vseh nevronih. Vseeno pa trenutni rezultati kažejo, da učinkovitost metode potrebuje nadgradnjo, z integracijo transgenov v genom. S tem bi rešili težave, ki nam jih povzroča mozaičnost. Inverzija se je izkazala zgolj delno uspešna, kjer vsekakor lahko potrdimo, da je do inverzije prišlo, ni pa rezultatov, ki bi kazali na to, da je bila popolna. To potrjuje tudi neuspešnost rešitve paralize, kjer smo sicer uspeli povrniti gibanje, ampak ne popolno. Tekom projekta smo odkrili številne pomanjkljivosti v tem pristopu, saj smo naleteli na številne nepričakovane komplikacije. Glavna planirana izboljšava je integracija transgenov v genom, ki bi potencialno zmanjšala variabilnost v izražanju gena, znotraj posameznih nevronov. Po drugi strani pa rezultati nakazujejo, da bi bilo morda bolje uporabiti kakšno drugo metodo, kot je Cre-Lox. Preden bo metoda nared za izvedbo željenih eksperimentov, bo potrebno še kar nekaj dela, ki bi odgovorili na vprašanja, ki smo si jih zadali na začetku projekta.
Sekundarne ključne besede:	C. elegans;nevronska dinamika;Cre-Lox;kloridni kanali;histamin;cognition;kognicija;
Vrsta datoteke:	application/pdf
Vrsta dela (COBISS):	Magistrsko delo/naloga
Komentar na gradivo:	University of Ljubljana, Joint interdisciplinary master's programme in Cognitive science in cooperation with Universität Wien, Sveučilište u Zagrebu, Univerzita Komenského v Bratislave and Eötvös Loránd Tudományegyetem
Strani:	52 str.
ID:	11029533