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The use of AAV vectors for transgene expression in primary afferent neurons in vitro and in vivo, and their utility in investigation of primary afferent anatomy and physiology

Student thesis: Doctoral ThesisDoctor of Philosophy

The aim of this project is to express various transgenic tools in adult mouse primary afferent neurons in vivo and use them to study various aspects of physiology and anatomy of primary afferent neurons. In order to achieve expression, we used non-pathogenic adeno-associated virus serotype 9 (AAV9). Our first aim was to establish an AAV delivery method that achieves high transduction in vivo in adult mouse primary afferents. We used AAV9 containing eGFP transgene in order to identify and characterise transduced neurons. We designed a new intrathecal delivery method that produces reliable and reproducible transduction in the majority of the L4 DRG neurons. After that, we investigated the impact various experimental parameters, such as viral titre and time post injection, have on the transduction pattern. 
After a successful delivery method was established, our next aim was to deliver functional transgenes to primary afferent neurons, such as GCaMP6s, in order to study activity of these neurons in vivo and in vitro. We assessed the performance of GCaMP6s in primary afferent cultures and attempted to establish an in vitro sensitisation model for calcium imaging that uses electrical stimulation as an activity trigger. Finally, we explored the use of our method of transgene delivery to express other transgenic tools in vivo, including Cre recombinase for the control of gene expression and an engineered glutamate-gated chloride channel for neuronal silencing.
Original languageEnglish
Awarding Institution
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Award date1 Jan 2019

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