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Striatal cholinergic cell assemblies in movement disorders

Periodic Reporting for period 5 - SynChI (Striatal cholinergic cell assemblies in movement disorders)

Reporting period: 2021-05-01 to 2021-10-31

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 focused on healthy control animals and at later stages of this action we sutdied 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.

After overcoming several technical setback (poor expression when using viral transfection, a faulty ChAT-Cre mouse that lead to ectopic expression when crossed with a reporter mouse) we completed the funding period with the data set we planned to attain: recordings from molecularly validated CINs in freely moving mice that perform self initiated and operantly conditioned actions. We also began to collect data from 6-OHDA treated mice before and after LID induction.These data will be analyzed and published in the future.
In the course of the project up until now we introduced two technologies into the lab: microendoscopic imaging in behaving mice (Aims 1 &3) and two-photon laser scanning microscopy (2PLSM) in brain slices (Aim 2). Both technologies were used to attain the goals of the project.
In terms of the microendocopy, the first period of the grant was spent establishing an SPF environment to conduct the experiments in the lab, purchasing and installation of the hardware and software and learning how to conduct the experiments. During the second period, we perfected the process of injecting GCaMP6f, a genetically encoded calcium indicators (GECI), into the striatum of ChAT-Cre mice and were able to get signals however the yield was very low because we apparently mostly over expressed the virus which either killed the cells or prevented them from providing a functional signal. In the third period, we attempted to fix this by conducting dilution studies, but in the end gave up on this and decided to acquire a newly available reporter mouse that expressed a Cre-dependent GECI in order to cross it with the ChAT-Cre mouse (jax 006410). In the 4th & 5th period we imaged the neural activity of cholinergic interneurons (CINs) in the crossed animals in both healthy, parkinsonian and dyskinetic mice and analyzed the data and prepared it for publication.
Unfortunately, in what we thought was a routine immunohistochemical (IHC) verification of the identity of the neuron’s molecular identity, we discovered in Oct. 2020 that the neurons we imaged all along were NOT cholinergic neurons. Having looked into this further we learned that several other labs (at Karolinska Institutet, New York University, etc) encountered this ectopic expression and abandoned the use of this mouse. As it turned out the investigator who contributed the mouse to jax discovered this issue and generated a new stock (jax 031661) which we promptly ordered. To begin the project from scratch. Period 4 thus ended with our establishing new colonies of crossed (corrected) Chat-Cre and Ai148 mice. Period 5 ended with the completion of the gathering of data from molecularly validated CINs in freely moving mice that perform self initiated and operantly conditioned actions, and have begun gathering similar data from 6-OHDA treated mice that will be treated with levodopa to induce dyskinesias.
We also identified the neurons that ectopically expressed GCaMP6f using the faulty ChAT-Cre mouse in order to possibly salvage the data for publication. We have found that the GCaMP6f expression neurons are mostly spiny projection neurons but also PV-fast spiking GABAergic interneurons. We are putting in final touches on a manuscript that discusses our conclusion from imaging a spare population of (mostly SPN) striatal neurons. We believe are insights are relevant for the interpretation of several high-profile publications about the statistical structure of cells assemblies that were derived from densely expressed neurons.

In terms of 2PLSM, this was used to produce publications about the nature of the GECI single observed in vivo in this mouse (Rehani et al. 2019) and excitability of CINs in parkinsonian mice (Tanimura et al 2016; Aceves et al 2019). We also are employing an optogenetic technique we developed to study the biophysics of nigral GABAergic neurons to estimate the distribution of active conductances in CIN dendrites. Thus, we expect to submit 3-4 manuscripts in 2022 that will result from the research conducted under this grant.
We will provide data concerning collective dynamics of cholinergic interneurons (ChIs) and possibly also serendipitously about GABAergic interneurons 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.
Monitoring collective neuronal activity in freely moving mice
Cholinergic neuropil activity are back-propagating action potentials
Upregulation of sodium currents in ChI dendrites boost cortical input in a mouse model of HD
multi photon microscopy of collective neuronal activity in acute brain slices
Amplification of NMDA current selectively in thalamic synapses onto ChIs in PD
Striatal TH expression before and after 6-hydroxdydopamine lesions to MFB