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...

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

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Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which glutamate or GABA are released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. Here we tested the hypothesis that the trans-synaptic cell adhesion molecules neurexins (Nrxns) regulate DA neuron neurotransmission. Conditional deletion of all Nrxns in DA neurons (DAT::Nrxns KO) revealed that loss of Nrxns does not impair the basic development and ultrastructural characteristics of DA neuron terminals. However, loss of Nrxns caused an impairment of DA transmission revealed as a reduced rate of DA reuptake following activity-dependent DA release, decreased DA transporter levels, increased vesicular monoamine transporter expression and impaired amphetamine-induced locomotor activity. Strikingly, electrophysiological recording revealed an increase of GABA co-release from DA neuron axons in the striatum of the KO mice. These findings reveal that Nrxns act as key regulators of DA neuron connectivity and DA-mediated functions.

High throughput quantitative analysis of microscopy images presents a challenge due to the complexity of the image content and the difficulty to retrieve precisely annotated datasets. In this paper we introduce a weakly-supervised MICRoscopy Analysis neural network (MICRA-Net) that can be trained on a simple main classification task using image-level annotations to solve multiple more complex auxiliary tasks, such as segmentation, detection, and enumeration. MICRA-Net relies on the latent information embedded within a trained model to achieve performances similar to state-of-the-art fully-supervised learning. This learnt information is extracted from the network using gradient class activation maps, which are combined to generate precise feature maps of the biological structures of interest. We demonstrate how MICRA-Net significantly alleviates the expert annotation process on various microscopy datasets and can be used for high-throughput quantitative analysis of microscopy images.