P 77 Optogenetic stimulations of striatal cholinergic interneurons in an animal model of dystonia

Abstract

Dystonias are movement disorders, defined by sustained or intermittent muscle contractions causing twisting movements and postures. The most prevalent inherited form of dystonia is caused by a mutation in the gene for torsin A (DYT1, ΔGAG) with incomplete penetrance. It has been hypothesized that an increased activity of cholinergic interneurons in the striatum, resulting in abnormal synaptic plasticity, plays an important role in the disease pathophysiology. However, this hypothesis is merely based on ex vivo electrophysiological recordings in brain slices of animal models which do not show a dystonic phenotype. Here we aim to substantiate the role of this alteration for motor dysfunctions in vivo by using optogenetic activation of striatal cholinergic interneurons in freely behaving mice. For this purpose we crossed Dyt1 ΔGAG heterozygous knock-in mice (DYT1) with mice expressing a light-dependent cation channel (Channelrhodopsin2, ChR2), essential for optogenetic examinations, specifically in cholinergic interneurons (Chat promotor). ChR2 is opened by blue light (wavelength 470 nm), leading to depolarization and acetylcholine release. The light (LED) is conveyed by optical cannulae, which were chronically implanted by stereotaxic surgery into the striatum of DYT1/ChR2 mice and wildtype (WT)/ChR2 littermates. First, we ensured that ChR2 is expressed in cholinergic interneurons of ChR2 + mice by immunohistochemistry and confocal microscopy and that these neurons respond to blue light exposure in brain slices. Subsequently, effects of different stimulation parameters (e.g. pulse width, Hz) on motor activity were examined in WT/ChR2 ( n  = 9) and DYT1/ChR2 mice ( n  = 6). Bilateral and unilateral photostimulations with blue LED light were compared to yellow LED light which does not open ChR2 (negative control). As expected, yellow light had no influence on behavior. Effects of bilateral stimulations with blue light were more robust than unilateral stimulations. Selected stimulation parameters induced significant increases of general activity (increased distance moved) specifically in DYT1/ChR2 mice compared to before or after stimulation, while there were no effects in WT/ChR2. This substantiates a specific role of striatal cholinergic neurons in motor activity in DYT1 dystonia. We have recently shown that DYT1 KI mice, which do not develop overt dystonia, present with sensorimotor deficits in specific behavioral paradigms. Optogenetic activation of cholinergic interneurons ameliorated this deficit temporarily. Preliminary data of an investigation of neuronal activity in the striatum after stimulation indicated an increased number of ChR2 cells positive for c-Fos in Dyt1/ChR2 ( n  = 4) in comparison to WT/ChR2 ( n  = 5) while there was no difference in total number of c-Fos positive cells. Total number of c-Fos positive cells did also not differ between naive WT/ChR2 and Dyt1/ChR2 mice, supporting that increased c-Fos expression in cholinergic interneurons is the expected effect of optogenetic stimulation on neuronal activity. We now perform comprehensive studies with short- and long-term stimulations in the Dyt1 mice to clarify the importance of overactive striatal cholinergic interneurons for the manifestation and abnormal striatal plasticity in DYT1 dystonia. For this purpose optogenetics will be combined with pharmacological, electrophysiological, neurochemical and molecular examinations.