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Chemical labelling for visualizing native AMPA receptors in live neurons

View Article: PubMed Central - PubMed

ABSTRACT

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

No MeSH data available.


Related in: MedlinePlus

Chemical labelling of native AMPARs in brain slices.(a) Western blot analyses of hippocampal slices after labelling using CAM reagents. Hippocampal slices were treated with 1 μM of CAM2(Fl) or CAM2(Ax488) in the absence or presence of 10 μM NBQX in ACSF. The cell lysates were analysed by western blotting using anti-Fl/OG, anti-Ax488, or anti-GluA2/3 antibody. (b) Confocal live imaging of labelled hippocampal slices with CAM2(Fl). To visualize neurons, hippocampal slices were acutely prepared from mice infected with lenti-virus encoding mCherry, and then treated with 1 μM of CAM2(Fl) in ACSF for 1 h. The composite Z-stack images of the live hippocampal slices are shown. Scale bars, 5 μm. In left, imaged region is shown as a magenta square. In upper right, zoomed overlay image is shown. Scale bars, 2 μm. (c) Immunostaining of labelled hippocampal slices with CAM2(Fl). Hippocampal slices treated with 1 μM of CAM2(Fl) in ACSF were fixed, permeabilized, and immunostained with anti-GluA2/3 antibody. Single plane confocal images of labelled slices immunostained with anti-GluA2/3 antibody are shown. Scale bars, 5 μm. (d) Single plane images (x–y) and ortho-images (x–z) of confocal Z-stacks of labelled hippocampal slices immunostained with anti-GluA2/3 antibody are shown. The yellow line indicates the region used for the x-z section. In the upper left, imaged region is shown as a magenta square. Closed or open arrow indicates top or bottom of the hippocampal slice, respectively. Scale bars, 100 μm. (e) Line profiling of Z-stack imaging shown in d. In top, comparison of line profiling of z-stack confocal imaging between labelled slice with CAM2(Fl) (n=12) and the slice immunostained with anti-GluA2/3 (n=13) is shown. In bottom, line profiling of the labelled slices obtained by confocal (n=12) or two-photon microscopy (n=15) is shown. Gradual fluorescent decrease dependent on the depth of the slice was observed by confocal imaging. In contrast, the fluorescent change was not observed in two-photon imaging. These results suggest that the fluorescent change was caused not by decrease of labelling efficiency but by low transparency of the excitation light.
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f4: Chemical labelling of native AMPARs in brain slices.(a) Western blot analyses of hippocampal slices after labelling using CAM reagents. Hippocampal slices were treated with 1 μM of CAM2(Fl) or CAM2(Ax488) in the absence or presence of 10 μM NBQX in ACSF. The cell lysates were analysed by western blotting using anti-Fl/OG, anti-Ax488, or anti-GluA2/3 antibody. (b) Confocal live imaging of labelled hippocampal slices with CAM2(Fl). To visualize neurons, hippocampal slices were acutely prepared from mice infected with lenti-virus encoding mCherry, and then treated with 1 μM of CAM2(Fl) in ACSF for 1 h. The composite Z-stack images of the live hippocampal slices are shown. Scale bars, 5 μm. In left, imaged region is shown as a magenta square. In upper right, zoomed overlay image is shown. Scale bars, 2 μm. (c) Immunostaining of labelled hippocampal slices with CAM2(Fl). Hippocampal slices treated with 1 μM of CAM2(Fl) in ACSF were fixed, permeabilized, and immunostained with anti-GluA2/3 antibody. Single plane confocal images of labelled slices immunostained with anti-GluA2/3 antibody are shown. Scale bars, 5 μm. (d) Single plane images (x–y) and ortho-images (x–z) of confocal Z-stacks of labelled hippocampal slices immunostained with anti-GluA2/3 antibody are shown. The yellow line indicates the region used for the x-z section. In the upper left, imaged region is shown as a magenta square. Closed or open arrow indicates top or bottom of the hippocampal slice, respectively. Scale bars, 100 μm. (e) Line profiling of Z-stack imaging shown in d. In top, comparison of line profiling of z-stack confocal imaging between labelled slice with CAM2(Fl) (n=12) and the slice immunostained with anti-GluA2/3 (n=13) is shown. In bottom, line profiling of the labelled slices obtained by confocal (n=12) or two-photon microscopy (n=15) is shown. Gradual fluorescent decrease dependent on the depth of the slice was observed by confocal imaging. In contrast, the fluorescent change was not observed in two-photon imaging. These results suggest that the fluorescent change was caused not by decrease of labelling efficiency but by low transparency of the excitation light.

Mentions: We subsequently examined whether CAM2 can successfully label AMPARs in their native three-dimensional environment in hippocampal and cerebellar tissue slices, which include a large number of glial cells (astrocytes, oligodendrocytes and microglial cells). Freshly prepared hippocampal slices (acute slices) were labelled with CAM2(Fl) or CAM2(Ax488) and evaluated by western blot analysis. As shown in Fig. 4a, a single strong band corresponding to AMPARs was observed in the presence of these labelling reagents, and this band disappeared by co-incubation with NBQX, indicating specific labelling of AMPARs in brain tissues.


Chemical labelling for visualizing native AMPA receptors in live neurons
Chemical labelling of native AMPARs in brain slices.(a) Western blot analyses of hippocampal slices after labelling using CAM reagents. Hippocampal slices were treated with 1 μM of CAM2(Fl) or CAM2(Ax488) in the absence or presence of 10 μM NBQX in ACSF. The cell lysates were analysed by western blotting using anti-Fl/OG, anti-Ax488, or anti-GluA2/3 antibody. (b) Confocal live imaging of labelled hippocampal slices with CAM2(Fl). To visualize neurons, hippocampal slices were acutely prepared from mice infected with lenti-virus encoding mCherry, and then treated with 1 μM of CAM2(Fl) in ACSF for 1 h. The composite Z-stack images of the live hippocampal slices are shown. Scale bars, 5 μm. In left, imaged region is shown as a magenta square. In upper right, zoomed overlay image is shown. Scale bars, 2 μm. (c) Immunostaining of labelled hippocampal slices with CAM2(Fl). Hippocampal slices treated with 1 μM of CAM2(Fl) in ACSF were fixed, permeabilized, and immunostained with anti-GluA2/3 antibody. Single plane confocal images of labelled slices immunostained with anti-GluA2/3 antibody are shown. Scale bars, 5 μm. (d) Single plane images (x–y) and ortho-images (x–z) of confocal Z-stacks of labelled hippocampal slices immunostained with anti-GluA2/3 antibody are shown. The yellow line indicates the region used for the x-z section. In the upper left, imaged region is shown as a magenta square. Closed or open arrow indicates top or bottom of the hippocampal slice, respectively. Scale bars, 100 μm. (e) Line profiling of Z-stack imaging shown in d. In top, comparison of line profiling of z-stack confocal imaging between labelled slice with CAM2(Fl) (n=12) and the slice immunostained with anti-GluA2/3 (n=13) is shown. In bottom, line profiling of the labelled slices obtained by confocal (n=12) or two-photon microscopy (n=15) is shown. Gradual fluorescent decrease dependent on the depth of the slice was observed by confocal imaging. In contrast, the fluorescent change was not observed in two-photon imaging. These results suggest that the fluorescent change was caused not by decrease of labelling efficiency but by low transparency of the excitation light.
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f4: Chemical labelling of native AMPARs in brain slices.(a) Western blot analyses of hippocampal slices after labelling using CAM reagents. Hippocampal slices were treated with 1 μM of CAM2(Fl) or CAM2(Ax488) in the absence or presence of 10 μM NBQX in ACSF. The cell lysates were analysed by western blotting using anti-Fl/OG, anti-Ax488, or anti-GluA2/3 antibody. (b) Confocal live imaging of labelled hippocampal slices with CAM2(Fl). To visualize neurons, hippocampal slices were acutely prepared from mice infected with lenti-virus encoding mCherry, and then treated with 1 μM of CAM2(Fl) in ACSF for 1 h. The composite Z-stack images of the live hippocampal slices are shown. Scale bars, 5 μm. In left, imaged region is shown as a magenta square. In upper right, zoomed overlay image is shown. Scale bars, 2 μm. (c) Immunostaining of labelled hippocampal slices with CAM2(Fl). Hippocampal slices treated with 1 μM of CAM2(Fl) in ACSF were fixed, permeabilized, and immunostained with anti-GluA2/3 antibody. Single plane confocal images of labelled slices immunostained with anti-GluA2/3 antibody are shown. Scale bars, 5 μm. (d) Single plane images (x–y) and ortho-images (x–z) of confocal Z-stacks of labelled hippocampal slices immunostained with anti-GluA2/3 antibody are shown. The yellow line indicates the region used for the x-z section. In the upper left, imaged region is shown as a magenta square. Closed or open arrow indicates top or bottom of the hippocampal slice, respectively. Scale bars, 100 μm. (e) Line profiling of Z-stack imaging shown in d. In top, comparison of line profiling of z-stack confocal imaging between labelled slice with CAM2(Fl) (n=12) and the slice immunostained with anti-GluA2/3 (n=13) is shown. In bottom, line profiling of the labelled slices obtained by confocal (n=12) or two-photon microscopy (n=15) is shown. Gradual fluorescent decrease dependent on the depth of the slice was observed by confocal imaging. In contrast, the fluorescent change was not observed in two-photon imaging. These results suggest that the fluorescent change was caused not by decrease of labelling efficiency but by low transparency of the excitation light.
Mentions: We subsequently examined whether CAM2 can successfully label AMPARs in their native three-dimensional environment in hippocampal and cerebellar tissue slices, which include a large number of glial cells (astrocytes, oligodendrocytes and microglial cells). Freshly prepared hippocampal slices (acute slices) were labelled with CAM2(Fl) or CAM2(Ax488) and evaluated by western blot analysis. As shown in Fig. 4a, a single strong band corresponding to AMPARs was observed in the presence of these labelling reagents, and this band disappeared by co-incubation with NBQX, indicating specific labelling of AMPARs in brain tissues.

View Article: PubMed Central - PubMed

ABSTRACT

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

No MeSH data available.


Related in: MedlinePlus