Étude de la capacité intrinsèque des neurones dopaminergiques à développer une connectivité non-synaptique

Dopamine (DA) neurons from the substantia nigra compacta (SNc) and ventral tegmental area (VTA) are key players of the neuronal circuitry regulating movement initiation, reward and learning. Their functioning and survival are also perturbed in diseases such as schizophrenia, drug abuse and Parkinson’s. The axonal connectivity of DA neurons is particularly intriguing due to the hyperdense nature of the axonal arbor of these neurons, containing a very large number of neurotransmitter release sites. Ultrastructural examination of the axon terminals established by DA neurons failed to identify a tight pre- and postsynaptic coupling at most of release sites, giving rise to the concept of non-synaptic terminals and “diffuse” or volume transmission. Furthermore, it is now well established that a subset of terminals established by DA terminals has the capacity to release other neurotransmitters such a glutamate and GABA.   A large literature implicates trans-synaptic proteins including neurexins (Nrxns) and neuroligins (Nlgns) in the development of synaptic contacts. In the presynaptic compartment, these cell adhesion molecules interact with active zone proteins like RIM1/2, Bassoon, ELKS and Munc-13 involved in regulating exocytosis. In the postsynaptic compartment, these cell adhesion molecules closely interact with scaffolding proteins like PSD95 or Gephyrin.  In this thesis work, we first performed an exhaustive characterization of axon terminals established by DA neurons in primary co-culture system. We evaluated the proportion and the molecular structure of synaptic and non-synaptic terminals established by VTA and SNc DA neurons. In our first article, using and efficient in vitro system, we demonstrated that DA neurons develop a small proportion of synaptic terminals that is strikingly lower compared to glutamatergic and GABAergic neurons. Interestingly, we discovered that the active zone protein Bassoon is mainly expressed in DA terminals that are in contact or in close proximity to a target cell, and less expressed in non-synaptic DA terminals. Finally, we found that overexpression of Nrxn-1SS4- in DA neurons leads to an increase in the proportion of synapses whereas, overexpression of Nlgn-1A+B is able to trigger a DA presynaptic differentiation of DA neurons, suggesting a key role for these transsynaptic proteins in synapse formation by DA neurons.   In a second article, we studied more globally the role of Nrxns proteins in the formation of synapses by DA neurons. We took advantage of a recently introduced triple conditional Nrxn mouse line to selectively delete Nrxns in DA neurons and examine the impact of this gene inactivation on the connectivity of DA neurons. In this part we demonstrated that the density of excitatory and inhibitory synapses density established by DA neurons is not affected by the deletion of all Nrxns, in comparison to the wild type group and does not impair the basic development and axonal connectivity of DA neurons at least, in vitro. In a second set of experiments, using patch-clamp recordings, we evaluated the function of excitatory and inhibitory synapses established by DA neurons in the striatum. GABA and glutamate synaptic currents evoked in the striatal medium spiny neurons by optogenetic stimulation of DA neuron axons revealed that glutamate release was unchanged, but identified a strong tendency for enhanced GABA co-release. Furthermore, using fast scan cyclic voltammetry, we found that the loss of Nrxns was associated with impaired DA transmission in the brain of adult mice, revealed by a reduced rate of DA reuptake after electrically-evoked DA release and with impaired amphetamine-induced locomotion.   With this thesis, we bring a new perspective on the capacity of DA neurons to develop an axonal arborization that is mainly non-synaptic. With this work, we provide strong evidence arguing that mesencephalic DA neurons are endowed with an intrinsic developmental program leading them to develop an axonal connectivity that is very different from striatal GABA neurons or cortical glutamate neurons. Ours findings suggest that although Nrxns and Nlgns are unlikely to be the main determinants of on the formation of synapses by DA neurons, that are likely to act as key regulators of DA and GABA signaling by these.

Keyphrases: Dopaminergic neurons, non-synaptic terminals, volume transmission, Neurexins, Neuroligins