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SynChI Report Summary

Project ID: 646880
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - SynChI (Striatal cholinergic cell assemblies in movement disorders)

Reporting period: 2015-05-01 to 2016-10-31

Summary of the context and overall objectives of the project

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 and Huntington's disease (another disorder that has been known 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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The first year and half have been dedicated to integrating two novel experimental methodologies – that are the backbone of our ambitious proposal - into our lab: two-photon laser scanning microscopy (2PLSM) and endoscopic in vivo imaging of genetically encoded calcium indicators (GECIs). The equipment – whose costs make up the lion’s share of the spending on this grant - was purchased immediately at the start of the grant (5/15) and both technologies are now totally functional and beginning to yield valuable data. Because of the complexity of both experimental set-up (technologically, experimentally, expertise-wise) this whole period was actually required to reach this point.
In the case of the endoscopic imaging the first stage was erecting an SPF (specific pathogen free) suite that handles stereotaxic viral transfection, animal instrumentation and endoscopic imaging in conjunction with a videoed 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 (attached please find an image depicting the monitoring of a mouse instrumented with and miniaturized endoscopic microscope within its behavior box alongside the signal detected from its brain). To reach this milestone, we also hosted an expert postdoc (Dr. Gabi Martins) from the Costa lab in Lisbon, who trained us. However, since that time point it took a full year to be able to generate a couple of animals that had good GECI signals and that learned the required behavior. We are not discouraged because we know that in order to get a usable animal many “stars need to be aligned”. Furthermore, when discussing the issues with other labs conducting endoscopic imaging and according to published manuscripts there is a 10-40% success rate in getting usable animals and we are certainly preforming within that range. In 8/16 I organized a workshop (IWoSC: International Workshop on Striatal Circuitry) with Profs. Pete Magill and Stephanie Cragg (Oxford University), and I travelled with two students to Oxford to present and get feedback about our preliminary findings from the endoscopic imaging experiments. The presentation of our data received very enthusiastic responses and we received valuable feedback on our work.
With regard to the 2PLSM, this too took time primarily because we needed to prepare a new space for the equipment which took a year. 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’ve had to work with them hand-in-hand for the next half year to resolve the issue and finally be able to run 2PLSM experiments in conjunction with optogenetics and electrophysiology towards the end of this reporting period. In the end, we have begun to collect very promising and interesting imaging data on the simultaneous ongoing activity in cholinergic interneurons in acute brain slices using 2PSLM and electrophysiology (see attached image that depicts ongoing calcium transients from 3 color coded active neurons imaged in acute brain slices).
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 have even generated data for a paper on Huntington’s disease (which is one of the diseases we promised to address in this DoA) that was accepted for publication a month after the end of the 1st reporting period. We have also developed our ability to successfully generate mouse toxin models of PD, which is a pre-requisite for our future PD related work (See figure of comparing TH staining in lesioned vs. nonleisioned animals).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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 both in awake, freely-moving mice and in reduced ex vivo preparations using 2PLSM. We are hopeful that the outcome of our studies will benefit fellow scientists, clinicians and patients that are involved in or affected by the debilitating movement disorders, whose etiology and progression we study in animal models.

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