Selective Silencing of Mammalian Neurons

The aim of this study is to continue developing a method for selective silencing of mammalian neurons using Glutamate-gated chloride channels. The neuroscience underlying diseases (neurological, psychiatric and drug abuse) can be understood best if the function of the various types of neurons in the central nervous system is known. This knowledge will help develop new therapies. Conventionally, function of neurons is investigated by inactivating them with pharmacological and lesion approaches. To overcome the disadvantages of these methods, we seek to continue developing a strategy to selectively silence neurons by using glutamate-gated chloride channels (GluCl). Invertebrate GluCl channels are introduced into mammalian neurons; their activation by the drug ivermectin inhibits firing of action potentials ("silencing"). These GluCl channels are composed of a heterooligomer of alpha- and beta-subunits. These GluCl channel subunits have been modified for our purposes: (i) They have been codon optimized for expression in mammalian cells; (ii) they have been tagged with different fluorescent proteins (cyan and yellow fluorescent protein) to allow for direct visualization. (iii) glutamate sensitivity has been reduced greatly by mutating Tyr182 to Phe in the beta- subunit (without affecting response to ivermectin). To achieve high expression levels in neurons we will insert the coding sequences into an adeno-associated virus-based system for gene transfer. In a first stage, we will infect isolated hippocampal neurons and test the concept of silencing by recording electrophysiological activities on isolated cells after application of ivermectin. If silencing succeeds in these cultures, these viruses will be injected stereotactically into the hippocampus of mice. Electrophysiological activities will be recorded on hippocampal cells of brain slices to show silencing in the context of an intact circuitry (analogous to the experiments in isolated cells). Additionally, we will apply standard tests for learning and memory formation in these injected animals as we expect deficits therein upon silencing of infected neurons in the hippocampus (Morris water maze, Lashley maze). The behavioral data can be correlated with electrophysiological results. A long-term objective of this project is the creation of animal models of neurological and psychiatric disorders. These transgenic mice will express GluCl channels in specified types of neurons with the help of cell-specific promoters.