Robustness of Excitability in Dopaminergic Neurons – ROBUSTEX

The methods used in this project are mainly electrophysiological methods that allow to determine the precise contribution of ion channel properties' variations to the observed variability in activity patterns. For instance, we use the action potential clamp technique to determine whether the variations in kinetics of the sodium and potassium currents activated during the action potential are responsible for the observed variability in shape and duration of the action potential from cell to cell. In addition, we use the dynamic clamp to simulate ionic conductances (IH, IA, and the Cav1.3-mediated calcium current) to determine whether the quantitative relationships between these currents (positive correlations, negative correlations, etc.) can explain the robustness of the electrical phenotype of dopaminergic neurons.

During the 18 months that have been funded by the ANR, we performed a characterization study that allowed us to define the electrical phenotype of dopaminergic neurons, in mature animals but also during postnatal development. This characterization was necessary before any attempt to define the robustness of the electrical phenotype. We used current-clamp recordings of numerous electrophysiological properties (16 different variables), and using multi-variate analyses (clustering, PCA) applied to these measurements, we were able to show that the mature electrical phenotype of these neurons is acquired by the end of the second postnatal week. Moreover, we showed that the developmental trajectory leading to this mature phenotype is non-linear. This study allowed us to propose a new method to precisely characterize the electrical phenotype of neurons, which we will use in the future to measure phenotypic variations induced by chronic perturbations of neuronal activity. This study was recently accepted for publication at eLife (minor revisions, mainly rewriting). In addition, concerning the optimization of action potential shape, we also performed a morphological characterization of dopaminergic neurons during development. We were able to show that the morphology of dopaminergic neurons, including axon location and axon initial segment (the site of action potential genesis) geometry were highly variable, even at early developmental stages. In spite of these morphological variations, the electrophysiological output of these neurons is rather stable. We are currently pursuing this study to determine whether the morphological variability has a significant impact on the energy expenditure related to action potential genesis and conduction. This work constituted the Master thesis of Estelle Moubarak, who has since obtained a Ministry PhD fellowship.

As mentioned before, the main perspectives of this project are to better define the principles underlying the robustness of activity in dopaminergic neurons, in order to understand why specific subpopulations are more resistant to neurodegeneration than others.

Dufour MA, Woodhouse A, Amendola J and Goaillard JM. Multi-dimensional analysis of electrical phenotype development in substantia nigra pars compacta dopaminergic neurons. (accepted at eLife with minor revisions).

Robustness is a general feature of complex evolvable biological systems. It is defined as the ability of a complex system to maintain function in the face of internal and external perturbations. The mammalian nervous sytem for instance is in some respect surprisingly robust to perturbations: post-lesional recovery or the preclinical asymptomatic phase of Parkinson’s disease are two expressions of this robustness. Ultimately, cognitive and behavioral robustness relies on the ability of single neurons to maintain information transfer constant. Unraveling the principles underlying the robustness of neuronal activity therefore seems critical to our understanding of the physiology and physiopathology of the nervous system.

In the current proposal, we will study the biophysical principles underlying robustness of neuronal activity in the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) of the mouse using electrophysiological and immunohistochemical approaches. We will particularly focus on how these neurons maintain a regular pacemaking activity and action potential shape and energy efficiency. To do so, we intend to study these firing properties in both unperturbed conditions (wild-type mice) and perturbed conditions (deletion of the gene for the Cav1.3 calcium channel). In particular, we want to determine the regulatory rules linking the Cav1.3 channel to the HCN and Kv4.3 channels. These three channels play major roles in the genesis of pacemaking activity in SNc DA neurons. In spite of its demonstrated role in pacemaking, the absence of the Cav1.3 channel does not significantly modify pacemaking, demonstrating remarkable compensation by other types of ion channels. Our personal data and the published literature suggest that a dynamic regulation of the properties of HCN and Kv4.3 channels is involved in this compensation.

Moreover, although pacemaking is maintained in the Cav1.3 KO mouse, it is unclear whether action potential shape is maintained. This is an important point since the action potential consumes a very significant part of the energy budget of neurons, and action potential shape has been related to its energy efficiency. Moreover, alterations in cell metabolism have been proposed to play a role in the neurodegeneration of specific subpopulations of SNc DA neurons. We will investigate whether the energy efficiency is maintained in the Cav1.3 KO mouse, but will also investigate whether SNc DA neurons all produce action potentials with similar energy efficiency in wild-type animals, or whether the energy efficiency of the action potential can be related to specific subpopulations of SNc DA neurons. In this part of the project, we will focus on the currents underlying the action potential to determine whether specific regulatory rules ensure that action potential shape and energy efficiency is maintained in the face of perturbations such as ion channel deletion or variation.

We believe that deciphering the rules underlying robustness of pacemaking and action potential shape in SNc DA neurons may have implications for our understanding of robustness of neuronal activity in general, but also for our understanding of the physiopathology of these neurons.