Optogenetično praćenje električne aktivnosti neurona primjenom fluorescentnih voltažnih senzora

master's thesis

• Title (english): Optogenetic monitoring of neuronal electrical activity using fluorescent protein-based voltage sensors
• Author: Željka Trepotec
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Dijana Škorić (član povjerenstva)
• Committee member: Duje Lisičić (član povjerenstva)
• Committee member: Inga Marijanović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2015-02-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Unatoč dosadašnjim postignućima u neuroznanosti, pitanja koja motiviraju znanstvenike o procesiranju informacija u neuronima još uvijek ostaju nerazjašnjena. Iako je proučavanje pojedinačnih neurona olakšano in vitro istraživanjima, ono iziskuje upotrebu neinvazivnih metoda visoke prostorne i vremenske rezolucije. Takva istraživanja omogućena su uvođenjem optogenetike kao novi eksperimentalni alat u neurofiziologiji. U ovom radu opisana je primjena genetički kodiranih fluorescentnih voltažnih senzora kao neinvazivno optogenetičko oruđe za bilježenje elektrofizioloških signala u podražljivim stanicama. U neurone korteksa uneseni su rekombinantni plazmidi hSyn_ArcLight i hSyn_Butterfly transdukcijom adeno – asociranim virusnim vektorima. Nakon ekspresije u neuronima, proteini su evaluirani elektrofiziološkim eksperimentima; električnom stimulacijom uz optičko bilježenje promjene fluorescencije. Time je dokazano da je ArcLight-A242 pogodno oruđe za bilježenje i detekciju pojedinačnih akcijskih potencijala. Iako bez značaja prema ovom istraživanju (zbog ograničenosti uređaja), Butterfly bi trebao biti još pouzdanije oruđe za detekciju, pa čak i promjena u potencijalu ispod praga okidanja. Uz ove rezultate, pretpostavka je da je optogenetika, obogaćena za ove dvije komponente, korak bliže neinvazivnom bilježenju električne aktivnosti neurona.

Abstract (english)

Despite all the recent achievements in the neuroscience, the questions that have motivated scientists about the information processing in individual neurons still remain unanswered. Studying neuronal behaviour in vitro facilitates the availability of a single neuron investigation, but requires non-invasiveness with high spatial and temporal resolution. Expansion of the neurophysiologic recording toolbox with the optogenetic stimulation has enabled such recordings. The included work shows the usage of genetically encoded voltage-sensitive fluorescent proteins as a non-invasive optogenetic tool for recording electrophysiological signals in electrogenic cells. The cortical neurons have been transduced with the new constructs, hSyn_ArcLight and hSyn_Butterfly by recombinant adeno-associated viral vectors. These novel constructs, specifically expressed in the neuronal cells, have been evaluated by electrophysiological experiments with electrical stimulations recorded optically. It has been shown that ArcLight A-242 is a valuable tool for recording and detection of single action potentials. Although not successful so far (due to the available equipment), Butterfly should be an even more reliable tool for the detection of even sub-threshold potential changes. Going forward, it might be assumed that the optogenetic toolbox, enriched by those two components, is a step closer to non-invasive, optical monitoring of neural electrical activity.

• Keywords: akcijski potencijal; elektrofiziologija; AAV transdukcija; molekularno kloniranje; patch- clamp; fluorimetrija
• Keywords (english): action potentials; electrophysiology; AAV transduction; molecular cloning; patch-clamp; Fluorometry
• Language: croatian
• URN:NBN: urn:nbn:hr:217:509850
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-04-24 09:01:09