Optogenetic augmentation of the hypercholinergic endophenotype in DYT1 knock-in mice induced erratic hyperactive movements but not dystonia

Abstract

Background The most prevalent inherited form of generalized dystonia is caused by a mutation in torsinA (DYT1, ∆GAG) with incomplete penetrance. Rodent models with mutated torsinA do not develop dystonic symptoms, but previous ex vivo studies indicated abnormal excitation of cholinergic interneurons (ChI) and increased striatal acetylcholine. Methods We used in vivo optogenetics to exacerbate this endophenotype in order to determine its capacity to trigger dystonic symptoms in freely behaving mice. Tor1a +/Δgag DYT1 mice and wildtype littermates expressing channelrhodopsin2 under the Chat promotor were implanted bilaterally with optical LED cannulae and stimulated with blue light pulses of varied durations. Findings Six months old DYT1 KI mice but not wildtype controls responded with hyperactivity to blue light specifically at 25 ms pulse duration, 10 Hz frequency. Neuronal activity (c-Fos) in cholinergic interneurons was increased immediately after light stimulation and persisted only in DYT1 KI over 15 min. Substance P was increased specifically in striosome compartments in naïve DYT1 KI mice compared to wildtype. Under optogenetic stimulation substance P increased in wildtype to match levels in Dyt1 KI, and acetylcholinesterase was elevated in the striatum of stimulated DYT1 KI. No signs of dystonic movements were observed under stimulation of up to one hour in both genotypes and age groups, and the sensorimotor deficit previously observed in 6 months old DYT1 KI mice persisted under stimulation. Interpretation Overall this supports an endophenotype of dysregulated cholinergic activity in DYT1 dystonia, but depolarizing cholinergic interneurons was not sufficient to induce overt dystonia in DYT1 KI mice.

TorsinB overexpression prevents abnormal twisting in DYT1 dystonia mouse models

“…TorsinB overexpression did not change cortical thickness, striatal volume, striatal Nissl+ small and medium cell number, or striatal GFAP immunoreactivity in Dlx-CKO and Cre control mice ( Figure S6A-D). TorsinB overexpression completely prevented the loss of dorsal striatal ChIs that have been linked to motor and electrophysiologic abnormalities in dystonia mouse models [23,[29][30][31][32][33][34]. Analysis using unbiased stereology demonstrated that Dlx-CKO mice had 33.5% fewer ChIs compared to Cre controls, whereas the B-OE allele completely prevented this loss ( Figure 4C-D).…”

Subtle changes in striatal muscarinic M1 and M4 receptor expression in the DYT1 knock-in mouse model of dystonia

“…In DYT1 knock-in (KI) mice, which do not develop dystonic symptoms like other viable DYT1 models [4], extracellular acetylcholine was found to be increased in the striatum and blocking of acetylcholine receptors normalized D2 receptor mediated effects on striatal ChI [5]. In addition to these interesting findings, our data on in vivo optogenetic stimulations of striatal ChI supported an endophenotype of dysregulated cholinergic activity, although depolarizing of these interneurons was not sufficient to induce overt dystonia in DYT1 KI mice [6].…”

“…They were bred and housed in the institute’s facility in groups up to 6 littermates. Genotypes were assessed by polymerase chain reaction (PCR) amplification analysis of DNA extracted from ear tissue using PuReTaq Ready-To-Go Beads (GE Healthcare) as described previously [6].…”

“…Tissue was processed for IHC based on previously published protocols [6, 14, 15]. Mice (n = 6/genotype) were deeply anesthetized with intraperitoneal injection of 100 mg/kg pentobarbital and perfused transcardially with 0.1 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS (Pfa).…”

Development of a fast liquid chromatography-tandem mass spectrometry method for simultaneous quantification of neurotransmitters in murine microdialysate

“…In brief, the microdialysis guide cannula was stereotactically implanted into the right murine striatum under 2.0% isoflurane anesthesia (CP-Pharma, Burgdorf, Germany) and 0.1% bupivacaine (Jenapharm, Jena, Germany) using the following coordinates in millimeter distance to the bregma and the skull surface according to the atlas of Franklin and Paxinos [17] in a stereotaxic frame (Stoelting, Wood Dale, IL, USA): anterioposterior + 0.8, mediolateral − 1.9, and dorsoventral 2.6. These coordinates were comparable to those used for previous experiments in mice [18]. The cannula was held in place with additional anchor screws and dental acrylic cement (Paladur ® , Heraeus Kulzer, Germany) on the skull surface.…”