Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice
| Autor: | Anton Dvorzhak, Rosemarie Grantyn |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Dendritic spine General Chemical Engineering Presynaptic Terminals Glutamic Acid Optogenetics General Biochemistry Genetics and Molecular Biology Synapse Mice 03 medical and health sciences Glutamatergic 0302 clinical medicine medicine Animals Active zone Pyramidal tracts General Immunology and Microbiology Chemistry General Neuroscience Glutamate receptor Brain Axons Huntington Disease Phenotype 030104 developmental biology medicine.anatomical_structure Astrocytes Synapses Artifacts Neuroscience 030217 neurology & neurosurgery Astrocyte |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/60113-v |
| Popis: | Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington's disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype. |
| Databáze: | OpenAIRE |
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