A nanothin block copolymer (BCP) brush-layer film adsorbed on glass nanofibers is shown to address the longstanding challenge of forming a template for the deposition of dense and well-dispersed nanoparticles on highly curved surfaces, allowing the development of an improved nanosensor for neurotransmitters. We employed a polystyrene-block-poly(4-vinylpyridine) BCP and plasmonic gold nanoparticles (AuNPs) of 52 nm in diameter for the fabrication of the nanosensor on pulled fibers with diameters down to 200 nm. The method is simple, using only solution processes and a plasma cleaning step. The templating of the AuNPs on the nanofiber surprisingly gave rise to more than 1 order of magnitude improvement in the surface-enhanced Raman scattering (SERS) performance for 4mercaptobenzoic acid compared to the same AuNPs aggregated on identical fibers without the use of a template. We hypothesize that a wavelength-scale lens formed by the nanofiber contributes to enhancing the SERS performance to the extent that it can melt the glass nanofiber under moderate laser power. We then show the capability of this nanosensor to detect the corelease of the neurotransmitters dopamine and glutamate from living mouse brain dopaminergic neurons with a sensitivity 1 order of magnitude greater than with aggregated AuNPs. The simplicity of fabrication and the far superior performance of the BCP-templated nanofiber demonstrates the potential of this method to efficiently pattern nanoparticles on highly curved surfaces and its application as molecular nanosensors for cell physiology.
Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.-Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.
Current electrophysiology and electrochemistry techniques have provided unprecedented understanding of neuronal activity. However, these techniques are suited to a small, albeit important, panel of neurotransmitters such as glutamate, GABA and dopamine, and these constitute only a subset of the broader range of neurotransmitters involved in brain chemistry. Surface-enhanced Raman scattering (SERS) provides a unique opportunity to detect a broader range of neurotransmitters in close proximity to neurons. Dynamic SERS (D-SERS) nanosensors based on patch-clamp-like nanopipettes decorated with gold nanoraspberries can be located accurately under a microscope using techniques analogous to those used in current electrophysiology or electrochemistry experiments. In this manuscript, we demonstrate that D-SERS can measure in a single experiment ATP, glutamate (glu), acetylcholine (ACh), GABA and dopamine (DA), among other neurotransmitters, with the potential for detecting a greater number of neurotransmitters. The SERS spectra of these neurotransmitters were identified with a barcoding data processing method and time series of the neurotransmitter levels were constructed. The D-SERS nanosensor was then located near cultured mouse dopaminergic neurons. The detection of neurotransmitters was performed in response to a series of K depolarisations, and allowed the detection of elevated levels of both ATP and dopamine. Control experiments were also performed near glial cells, showing only very low basal detection neurotransmitter events. This paper demonstrates the potential of D-SERS to detect neurotransmitter secretion events near living neurons, but also constitutes a strong proof-of-concept for the broad application of SERS to the detection of secretion events by neurons or other cell types in order to study normal or pathological cell functions.
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...
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