Final Report Summary - DEEPNMDAR (DEEP BRAIN TISSUE IMAGING OF GLUTAMATE NMDAR)
Unrevealing the rules that govern the surface trafficking and membrane stabilization of neurotransmitter receptors, in particular the N-methyl-D-aspartate receptor (NMDAR) is crucial for our understanding of several physiopathological brain processes. Over the past few years, scientists have shown that neurotransmitter receptors diffuse inside and out of synapses in cultured neurons using single molecule approaches. These findings drastically changed the way we understand synaptic transmission and adaptation.
The finalised project aimed to study the surface diffusion of NMDAR in intact hippocampal slices, providing the first functional map of such key receptors in native neuronal networks. Due to the difficulties of imaging single molecules in high background noise tissue, a multidisciplinary approach has been adopted using quantum dots and single walled nanotubes to track single NMDAR neurotransmitters.
Since the beginning of the project, different kind of nanoparticles have been tested in order to explore the possibilities of performing single molecule tracking techniques in the brain. Quantum dots, organic self-assembled nanoparticles, and carbon nanotubes have been optically studied and characterized, successfully detecting single nano-objects in deep brain tissue. The interaction of several synthesis and chemical modifications of these three types of nanoparticles with mammalian cells and in particular with brain cells were thoroughly studied, ruling out nanoparticles that presented toxic effects.
Non-toxic nanoparticles with good optical properties were selected, and a characterization of the diffusion of nanoparticles in the brain has been performed in multiple time and space scales, optimizing the conditions needed to perform single receptor tracking in acute brain slices.
Once the ground work regarding detection of single nanoparticles in the brain was finished, functionalized nanoparticles were injected in animals aiming to target single neuronal receptors. We first focused on Dopamine receptors, as they are very mobile and responsible of the detection of one of the most important neuromodulators in the brain. We succeeded in expressing dopamine receptors with an extracellular univocal tag in rats, to which we targeted functionalized quantum dots. In this way, single neuronal receptors could be successfully tracked in acute brain slices for the first time to our knowledge.
In parallel, single walled carbon nanotubes were imaged in rat brain slices, testing the potential of these nanoprobes for neurophotonic applications. We successfully tracked individual nanotubes in acute brain slices, characterizing the extracellular space of a living brain slice at a spatial resolution that has never been achieved before.
The main goal of the project was to track single NMDAR in acute slices. We successfully expressed engineered NMDAR in rat hippocampus and used near infra-red emitting quantum dots to track them in acute slices. This final stage of the project was successfully accomplished, providing for the first time a characterization of NMDAR dynamics in intact brain tissue.
The finalised project aimed to study the surface diffusion of NMDAR in intact hippocampal slices, providing the first functional map of such key receptors in native neuronal networks. Due to the difficulties of imaging single molecules in high background noise tissue, a multidisciplinary approach has been adopted using quantum dots and single walled nanotubes to track single NMDAR neurotransmitters.
Since the beginning of the project, different kind of nanoparticles have been tested in order to explore the possibilities of performing single molecule tracking techniques in the brain. Quantum dots, organic self-assembled nanoparticles, and carbon nanotubes have been optically studied and characterized, successfully detecting single nano-objects in deep brain tissue. The interaction of several synthesis and chemical modifications of these three types of nanoparticles with mammalian cells and in particular with brain cells were thoroughly studied, ruling out nanoparticles that presented toxic effects.
Non-toxic nanoparticles with good optical properties were selected, and a characterization of the diffusion of nanoparticles in the brain has been performed in multiple time and space scales, optimizing the conditions needed to perform single receptor tracking in acute brain slices.
Once the ground work regarding detection of single nanoparticles in the brain was finished, functionalized nanoparticles were injected in animals aiming to target single neuronal receptors. We first focused on Dopamine receptors, as they are very mobile and responsible of the detection of one of the most important neuromodulators in the brain. We succeeded in expressing dopamine receptors with an extracellular univocal tag in rats, to which we targeted functionalized quantum dots. In this way, single neuronal receptors could be successfully tracked in acute brain slices for the first time to our knowledge.
In parallel, single walled carbon nanotubes were imaged in rat brain slices, testing the potential of these nanoprobes for neurophotonic applications. We successfully tracked individual nanotubes in acute brain slices, characterizing the extracellular space of a living brain slice at a spatial resolution that has never been achieved before.
The main goal of the project was to track single NMDAR in acute slices. We successfully expressed engineered NMDAR in rat hippocampus and used near infra-red emitting quantum dots to track them in acute slices. This final stage of the project was successfully accomplished, providing for the first time a characterization of NMDAR dynamics in intact brain tissue.