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Striatal cholinergic cell assemblies in movement disorders

Periodic Reporting for period 3 - SynChI (Striatal cholinergic cell assemblies in movement disorders)

Reporting period: 2018-05-01 to 2019-10-31

Pathological neuronal synchrony is the hallmark of many neurological disorders, including Parkinson's disease (PD), epilepsy, dystonia, tremors, autism and schizophrenia and possibly others, as well. However, standard methodologies using microelectrode recordings to monitor the collective (and possible synchronous) activity of neurons in animal models of these diseases typically do not enable the simultaneous monitoring of more that a handful of neurons. Moreover, even methodologies that push this limit cannot guarantee the specific neuronal types of neurons recorded. This latter constraint is paramount because many neurological disorders are typically characterized by an insult that is selective to specific neuronal types, and therefore it is critical to be able to guarantee that one can study exactly those neurons.
In order to overcome these constraints, we opted to use optical imaging of neurons that we can selectively target (using genetic manipulations) to express calcium indicators in our cell type of interest - which is the cholinergic interneuron of the striatum. The receptors activated by the output of these interneurons are therapeutically important in PD, as they were the target of the first available anticholinergic treatment of PD, prior to the advent of dopamine replacement therapy (DRT). Anticholinergics were very effective and are sometimes still used today particularly on young tremulous PD patients.
Thus, our overall objectives of the action is to image molecularly-identified cholinergic interneurons using endoscopic imaging that enables the monitoring of large assemblies of these cells in awake, freely-moving mice. We are initially focusing on healthy control animals but eventually at later stages of this action we intend to also study mouse models of Parkinson's disease with an emphasis on levodopa induced dyskinesia (that has recently been shown to be accompanied by dysregulation of cholinergic signaling in the striatum). We believe that understanding the collective dynamics of cholinergic interneurons in healthy and diseased mice will lead to novel insights into the disease mechanisms and could potentially identify additional therapeutic targets.
The first year and half was spent integrating two novel experimental methodologies – that are the backbone of our ambitious proposal - into our lab: two-photon laser scanning microscopy (2PLSM) and endoscopic imaging of genetically encoded calcium indicators (GECIs) in freely moving mice. The equipment was purchased immediately at the start of the grant (5/15) and both technologies are now functional and yielding valuable data. Because of the complexity of both experimental set-up (technologically, experimentally, expertise-wise) spending 18 months on this was justifiable, as explained presently.
In the case of the endoscopic imaging (for the purpose of Aims 1c and 3 of the DoA) the first stage was erecting an SPF (specific pathogen free) suite in which we conduct stereotaxic viral transfection, animal instrumentation and endoscopic imaging within a videoed and IR-illuminated behaviour box. Building the setup took a few months and by the half year mark we already had an instrumented animal that yielded beautiful calcium signals. However, since that time point it has unfortunately taken two full years to “align all the stars” to get these experiments to work (due mainly to problems with transfection). Nevertheless, our preliminary data immediately yielded an unexpected finding that actually contradicts the dogmatically held view according to which the discharge of ChIs is not related to movement per se. This view is based on experiments that were conducted since the 1980s in highly-trained and head-restrained primates. We believe that our data tell a different story because our animals are freely moving and most of their movements are self-initiated movements that are either highly stereotypical (and innate) like grooming or exploratory. Both of these are very different from the highly-trained and artificially-learned movements that the primates made in order to receive reward. We will therefore be focusing on this novel and exciting finding in the next year, and believe that they will yield a high-profile publication.
With regard to the 2PLSM, it took a full year to be allotted and prepare the new space for the equipment. The equipment was finally set up in 5/16, and because we had negotiated with the supplier (Femtonics, Budapest) to integrate optogenetics and electrophysiology into their 2PLSM system, we had to work with them hand-in-hand for the next half year to resolve several technical issues and finally be able to run 2PLSM experiments in conjunction with optogenetics about a year ago. Since then we have completed the 2PLSM acute slice study that we outlined in Aims 1a and 2 of the DOA, namely imaging small networks of striatal cholinergic interneurons (ChIs) expressing the GECI GCaMP6s in acute slices while activating thalamic afferents optogenetically. While this project yielded valuable data to help better interpret the signal recorded in the endoscopic imaging in the freely moving animals, we've had only a partial success in characterizing the collective activity of ChIs that could be visualized with the GCaMP6s (even though it was often possible to visualize 3-10 cells per slice).
Throughout the whole period, we produced data related to the grant using the equipment in the lab (standard electrophysiology in conjunction with optogenetic and wide field imaging) and published two papers one adaptations in ChIs in Huntington’s disease (Tanimura et al. 2016) and in Parkinson’s disease (Aceves-Buendia et al. 2017), which are the diseases we promised to address in this DoA. For the purpose of this latter paper, we developed our ability to successfully generate dopamine depleted mice using 6-OHDA, and have begun testing protocols for levodopa induced dyskinesias (LIDs) which are necessary for our future PD related work. While LIDs per se were not the focus of the original proposal, since then there have been a battery of published papers about the central role of ChIs in LIDs. We therefore feel that it would b
As mentioned above we have already begun to yield completely novel (and beyond the state-of-the-art) data concerning collective dynamics of cholinergic interneurons (ChIs) both in awake, freely-moving mice and in reduced ex vivo preparations using 2PLSM. This data counter the dogma in the field and show for the first time that the collective activity of ChIs is strongly modulated during exploratory, self-initiated movement. We hope by the end of the grant period to elucidate this novel role of ChIs. Additionally, we will study the how ChI assemblies evolve as the mice undergo classical conditioning followed by extinction with the hope of revealing how collective activity of ChIs encodes this learning process. Finally, we will elucidate how this network is altered in dopamine depleted mice both before and after inducation of levodopa-induced dyskinesias. This endoscopic imaging studies of Chis will continue to be complemented by 2PLSM experiments in acute slices to better understand the biophysical adaptations that occur in the ChIs in these diseased conditions. We are hopeful that the outcome of our studies will benefit fellow scientists, clinicians and patients that are involved in or affected by these debilitating movement disorders.
multi photon microscopy of collective neuronal activity in acute brain slices
Striatal TH expression before and after 6-hydroxdydopamine lesions to MFB
Upregulation of sodium currents in ChI dendrites boost cortical input in a mouse model of HD
Monitoring collective neuronal activity in freely moving mice
Amplification of NMDA current selectively in thalamic synapses onto ChIs in PD
Cholinergic neuropil activity are back-propagating action potentials