Electrophysiological mapping of networks dynamics in basal ganglia in normal and Parkinsonian conditions using optogenetic control of specific neuronal pathway. – OPTOMAP-Parkin

Our research proposal takes full advantage of recently developed techniques combining genetics and optics that can assess for causal relationship. Such optogenetic methods rely on the use of modified adeno-associated virus (AAV) to express light-sensitive protein called opsins into neurons of interest. One great advantage of such optogenetic strategy is to be able to manipulate specific inputs. Indeed, only the cells infected by the virus will respond to the laser stimulation. The cell-specificity of this approach is also provided by the sequence used as a promoter in the viral construction and the site of viral injection. Here, we principally use AAV controlled by a hSynapsin promoter to target inhibitory GP neurons (see illustration). Different opsins are used depending on the desired effect: excitation or inhibition. We are using Channelrhodopsin-2 (ChR2), a nonspecific cations channel, for depolarizing neurons and, Archaerhodopsins-3 (hSyn-eArch3.0 or CaMKIIa-eArchT3.0), a light-driven ion pump which extrude proton to silence neurons. Importantly, optogenetic manipulation has unprecedented time-scale resolution and switching cells ‘on’ or ‘off’ occurs within milliseconds. More recently, we have also started to take advantage of novel transgenic mice line expressing the protein recombinase Cre in specific subpopulation of neurons, thus providing additional cellular specificity for our optogenetic manipulation.

Our preliminary experiments, combining optogenetic manipulation of all GPe neurons and electrophysiological recordings, establish the fundamental principle that the GPe is necessary and sufficient for the maintenance and the generation of abnormal beta oscillations (see illustration). Our optogenetic manipulations of GPe neurons also established the fundamental principle that GPe neurons control the firing rate and synchronization level within the whole basal ganglia circuits. These important results partly contradict classic models of basal ganglia functional organization that traditionally consider GPe only as a relay nucleus. We are now going one step further into the mechanistic understanding of abnormal beta oscillations generation by directly dissecting the specific and causal contribution of the two main population of GPe neurons (the so-called Proto and Arky neurons). Our preliminary data have been presented multiple times at local (synapse day 2016, Bordeaux) and international meetings (German Neuroscience meeting, Goettingen 2015).

Most past studies have focused on the neuronal changes that correlate with Parkinson’s disease and, as such, it is difficult to apprehend if those changes are the consequence or the cause of those disorders. In this research proposal, we propose to study the importance of synchronized oscillations in basal ganglia and will use novel optogenetic methods to go beyond correlation and test for causality. Our future experiments will be focused on defining the functional importance of Proto and Arky neurons in the generation and maintenance of abnormal beta oscillations and their consequence on motor behavior. We will thus provide new mechanistic insights to better control these abnormal neural events.

One key aspect of OPTOMAP-Parkin is to understand the role of synchronized oscillatory activity during motor behavior. The funding provided by the ANR gave us the opportunity to develop a new experimental paradigm: electrophysiological recordings in awake head-fixed animals. The first results obtained with this newly-developed head-fixed preparation has recently been published (Mallet et al., 2016 Neuron, doi:10.1016/j.neuron.2015.12.017). This new method will now be implemented in rats trained to perform a lever press motor task that will be suitable to directly tackle our questions of interest.

The basal ganglia circuits form a complex loop of nuclei that connect the cortex to the thalamus and are critically important for motor and cognitive behaviors. During motor behaviors, basal ganglia circuits are involved in many aspects such as the selection and the initiation of motor plans but also the suppression of unwanted actions. The importance of basal ganglia circuits in movement is perhaps best illustrated by the consequences of their dysfunction and the devastating motor impairments that appear following the dopamine loss occurring in Parkinson Disease (PD). Akinesia/bradykinesia is one of the main symptoms in unmedicated PD and it describes the inability to start/the slowing down of movement. Furthermore, prolonged levodopa (L-DOPA) therapy, the ‘gold standard’ treatments for PD, inevitability leads to unbearable side effects which are the exact opposite of PD, that is a paradoxical hyperkinetic behavior characterized by frequent abnormal and involuntary movements also known as L-DOPA induced dyskinesias (LIDs). These LIDs cannot be suppressed voluntary and are very debilitating for patients. Altogether, in PD, both movement initiation and cancellation processes can be altered at different time course of the disease but which of the multiple basal ganglia neuronal pathways directly underlie those alterations is not known.
Specialized electrical activities known as ‘synchronized oscillations’ are used for network dynamics and are important for optimal communication in healthy brain circuits. Oscillations can be distinguished by their characteristic frequencies. In both humans and animals, the emergence of distinct oscillations is correlated with different motor outcomes. For example, in the healthy brain motor system, the so-called ‘beta’ oscillations (15-30 Hz) accompany the maintenance of postural sets and are linked with ‘anti-movement’ functions. Conversely, ‘gamma’ oscillations (40-80 Hz) are associated with the initiation of new motor actions, linking them to ‘pro-movement’ functions. Importantly though, if synchronized oscillations are not properly controlled in space and time, they could become counterproductive or truly pathological. Indeed, excessive synchronization of neuronal activity is recognized as a critical functional change accompanying Parkinsonism. Interestingly, different frequencies have been associated with the different symptoms of PD. More specifically, excessively synchronized oscillations at beta frequencies have been associated with akinesia, whereas pronounced gamma oscillations expression has been correlated to the paradoxical hyperkinetic behavior visible during the LIDs. Therefore, abnormally synchronized oscillations might be responsible for both hypokinetic and hyperkinetic behaviors in PD. With this in mind, it is now especially timely and important to go beyond correlational analysis. Here, using newly developed method, I will dissect the causal relationship between basal ganglia circuits and their involvement in abnormal motor behavior of rodents. I will employ a multidisciplinary approach combining cutting edge optogenetic toolboxes, electrophysiology, and behavior to investigate how targeted neuronal pathway causally influence network dynamic in basal ganglia and behavior. Altogether, this strategy will give us valuable insights into the neuronal processes that underlie ‘Start’ and ‘Stop’ signals within BG.
The goal and ambition of this project is to provide a critical step towards the identification of new candidate therapies that can be used to reduce the severity of PD and LIDs. Hence, my research proposal will be at the interface of basic neuroscience and clinical research, with the ultimate goal of translating discoveries from bench to bedside. Furthermore, this study will greatly increase our understanding of how basal ganglia and their partner circuits work together to influence behavior.