Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Rare genetic mutations in genes such as Parkin, Pink1, DJ-1, α-synuclein, LRRK2 and GBA are found to be responsible for the disease in about 15% of the cases. A key unanswered question in PD pathophysiology is why would these mutations, impacting basic cellular processes such as mitochondrial function and neurotransmission, lead to selective degeneration of SNc DA neurons? We previously showed in vitro that SNc DA neurons have an extremely high rate of mitochondrial oxidative phosphorylation and ATP production, characteristics that appear to be the result of their highly complex axonal arborization. To test the hypothesis in vivo that axon arborization size is a key determinant of vulnerability, we selectively labeled SNc or VTA DA neurons using floxed YFP viral injections in DAT-cre mice and showed that SNc DA neurons have a much more arborized axon than those of the VTA. To further enhance this difference, which may represent a limiting factor in the basal vulnerability of these neurons, we selectively deleted in mice the DA D2 receptor (D2-cKO), a key negative regulator of the axonal arbour of DA neurons. In these mice, SNc DA neurons have a 2-fold larger axonal arborization, release less DA and are more vulnerable to a 6-OHDA lesion, but not to α-synuclein overexpression when compared to control SNc DA neurons. This work adds to the accumulating evidence that the axonal arborization size of SNc DA neurons plays a key role in their vulnerability in the context of PD.

In Parkinson's disease, the most vulnerable neurons are found in the ventral tier of the substantia nigra (SN), while the adjacent dopamine (DA) neurons of the ventral tegmental area (VTA) are mostly spared. Although a significant subset of adult VTA DA neurons expresses Vglut2, a vesicular glutamate transporter, and release glutamate as a second neurotransmitter in the striatum, only very few adult SN DA neurons have this capacity. Previous work has demonstrated that lesions created by neurotoxins such as MPTP and 6-hydroxydopamine (6-OHDA) can upregulate the expression of Vglut2 in surviving DA neurons.Currently, the molecular mechanisms explaining the plasticity of Vglut2 expression in DA neurons are unknown, as are the physiological consequences for DA neuron function and survival. Here we aimed to characterize the developmental expression pattern of Vglut2 in DA neurons and the role of this transporter in post-lesional plasticity in these neurons. Using an intersectional genetic lineage-mapping approach, based on Vglut2-Cre and TH-Flpo drivers, we first found that more than 98% of DA neurons expressed Vglut2 at some point in their embryonic development. Expression of this transporter was detectable in most DA neurons until E11.5 and was found to be localized in developing axons. Moderate enhancement of VGLUT2 expression in primary DA neurons caused an increase in axonal arborization length. Compatible with a developmental role, constitutive deletion of Vglut2 caused a regional defect in TH-innervation of the dorsal striatum in E18.5 embryos. Moreover, using an in vitro neurotoxin model, we demonstrate that Vglut2 expression can be upregulated in post-lesional DA neurons by 2.5-fold, arguing that the developmental expression of Vglut2 in DA neurons can be reactivated at postnatal stages and contribute to post-lesional plasticity of dopaminergic axons. In support of this hypothesis, we find fewer mesostriatial dopaminergic projections in the striatum of conditional Vglut2 KO mice 7 weeks after a neurotoxic lesion, compared to control animals. Thus, we propose here that one of the functions of Vglut2 in adult DA neurons is to promote post-lesional recovery of meso-striatal axons.

30Chemical neurotransmission in the brain typically occurs through synapses, which are structurally 31 and functionally defined as sites of close apposition between an axon terminal and a postsynaptic 32 domain. Ultrastructural examinations of axon terminals established by monoamine neurons in 33 the brain often failed to identify a similar tight pre-and postsynaptic coupling, giving rise to the 34 concept of "diffuse" or "volume" transmission. Whether this results from intrinsic properties of 35 such modulatory neurons remains undefined. Using an efficient co-culture model, we find that 36 dopaminergic neurons establish an axonal arbor that is distinctive compared to glutamatergic or 37 GABAergic neurons in both size and propensity of terminals to avoid direct contact with target 38 neurons. Furthermore, while most dopaminergic varicosities express key proteins involved in 39 exocytosis such as synaptotagmin 1, only 20% of these are synaptic. The active zone protein 40 bassoon was found to be enriched in a subset of dopaminergic terminals that are in proximity to 41 a target cell. Irrespective of their structure, a majority of dopaminergic terminals were found to 42 be active. Finally, we found that the presynaptic protein Nrxn-1 SS4and the postsynaptic protein 43 NL-1 AB , two major components involved in excitatory synapse formation, play a critical role in the 44 formation of synapses by dopamine neurons. Taken together, our findings support the idea that 45 dopamine neurons in the brain are endowed with a distinctive developmental program that leads 46 them to adopt a fundamentally different mode of connectivity, compared to glutamatergic and 47 GABAergic neurons involved in fast point-to-point signaling. 48 49 51 SIGNIFICANCE STATEMENT 52Midbrain dopamine (DA) neurons regulate circuits controlling movement, motivation, and 53 learning. The axonal connectivity of DA neurons is intriguing due to its hyperdense nature, with a 54 particularly large number of release sites, most of which not adopting a classical synaptic 55 structure. In this study, we provide new evidence highlighting the unique ability of DA neurons to 56 establish a large and heterogeneous axonal arbor with terminals that, in striking contrast with 57 glutamate and GABA neurons, actively avoid contact with the target cells. The majority of synaptic 58 and non-synaptic terminals express proteins for exocytosis and are active. Finally, our finding 59 suggests that, NL-1 A+B and Nrxn-1 SS4-, play a critical role in the formation of synapses by DA 60 neurons. 61 62 postsynaptic coupling at most release sites, giving rise to the concept of "diffuse" or "volume" 85 transmission, whereby neurotransmitter release from non-synaptic axon terminals leads to 86 activation of metabotropic receptors on target cells located at a distance, within a sphere of a 87 few tens of microns (17)(18)(19)(20)(21)(22)(23)(24). 88The molecular mechanisms determining the ability of DA neurons to establish synaptic 89 and non-synaptic terminals are presently unknown. M...

No abstract

Chemical neurotransmission typically occurs through synapses. Previous ultrastructural examinations of monoamine neuron axon terminals often failed to identify a pre-and postsynaptic coupling, leading to the concept of "volume" transmission. Whether this results from intrinsic properties of these neurons remains undefined. We find that dopaminergic neurons in vitro establish a distinctive axonal arbor compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons.While most dopaminergic varicosities are active and contain exocytosis proteins like synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in dopaminergic terminals that are in proximity to a target cell. Finally, we found that the proteins neurexin-1α SS4− and neuroligin-1 A+B play a critical role in the formation of synapses by dopamine

Dopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment thought a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity, is stable in response to electrical but not optogenetic train pulses and shows a calcium sensitivity that is comparable to that of axonal release. It is also strikingly more resilient compared to axonal release in a 6‐ hydroxydopamine model of Parkinson’s disease plasticity. We find that the molecular mechanism of STD DA release differs from axonal release with regards to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knock-out Syt4 and Syt7 is not sufficient to reduce STD DA release, removal of both isoforms reduces this release by ~50%, leaving axonal release unimpaired. Our works unveils clear differences in the mechanisms of STD and axonal DA release.

Dopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment through a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity and shows a calcium sensitivity that is comparable to that of axonal release. We find that the molecular mechanism of STD DA release differs from axonal release with regard to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knockout of Syt4 or Syt7 is not sufficient to reduce STD DA release, the removal of both isoforms reduces this release by approximately 50%, leaving axonal release unimpaired. Our work unveils clear differences in the mechanisms of STD and axonal DA release.

Midbrain dopamine (DA) neurons, a population of cells that are critical for motor control, motivated behaviors and cognition, release DA via an exocytotic mechanism from both their axonal terminals and their somatodendritic (STD) compartment. In Parkinson's disease (PD), it is striking that motor dysfunctions only become apparent after extensive loss of DA innervation. Although it has been hypothesized that this resilience is due to the ability of many motor behaviors to be sustained through a basal tone of DA and diffuse transmission, experimental evidence for this is limited. Here we conditionally deleted the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (cKODA mice) to abrogate most activity-dependent axonal DA release in the striatum and mesencephalon, leaving STD DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks, suggesting that activity-dependent DA release is dispensable for such basic motor functions. Basal extracellular levels of DA in the striatum were unchanged, suggesting that a basal tone of extracellular DA is sufficient to sustain basic movement. We also found multiple adaptations in the DA system of cKODA mice, similar to those happening at early stages of PD. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.