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Abstract:
So far, various methods for reversible silencing of neuronal networks in mouse brains in vivo have been described in the literature. Besides their theoretical usefulness for exploring the relationship between altered function of specifc neuronal circuits in the brain and behavioral phenotypes that emerge from these altered network dynamics, many practical pitfalls have to be overcome.
In 2007, David J. Anderson described a method that made use of a viral overexpressing system of a heteromeric, codon-optimized, chloride channel from C. elegans (GluClaß) that could be gated by i.p. application of the drug lvermectin (IVM)- a weil known anthelmintic drug - and thereby elicited reversible silencing in mouse brain neurons (Lerchner et al. Reversible silencing of neuronal excitability in behaving mice by a genetically targeted, ivermectin-gated Cl- channel. Neuron (2007) vof. 54 (1) pp. 35-49).
In 2010, W. Lynch explored the possibilities of creating a humanized, monomeric glycine receptor with improved gating capabilities in response to IVM (Lynagh und Lynch. An lmproved lvermectin-activated Chloride Channel Receptor for fnhibiting Electrical Activity in Defined Neuronal Populations. Journal of Biological Chemistry (2010) vol. 285 (20) pp. 14890-14897). His construct consists of the human glycine receptor alpha 1 subunit to which two point mutations were introduced: The first mutation (F207 A) eliminates the Glycine sensitivity of the receptor in expression systems in vitro, the second mutation (A288G) increases sensitivity to ivermectin in vitro almost 100-fold to application on a nanomolar scale.
Motivated by his findings we tried to implement this construct into an rAAVexpression system which then could be used for further in vitro andin vivo studies.
Our primary goal was to observe expression of the construct in vitro (primary neuron cultures) and then further explore its dynamics in two behavioral paradigms.
