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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.


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Chemical labelling of native AMPARs in cultured neurons.(a) Western blot analyses of cultured neurons after labelling using CAM reagents. Cultured cortical neurons were treated with 1 μM of CAM2(OG), CAM2(Fl), CAM2(Ax488), or CAM2(Bt) in the absence or presence of 10 μM NBQX in serum free Neurobasal medium. The cell lysates were analyzed by western blot using anti-Fl/OG, anti-Ax488, or anti-GluA2 antibody, or by biotin blotting using streptavidin-HRP. * indicates biotinylated proteins endogenously expressed in the neurons. (b) Effect of competitive antagonists for glutamate receptors on chemical labelling of native AMPARs in cultured neurons. Western blot analyses of cultured neurons after labelling using CAM reagents are shown. Cultured cortical neurons were treated with 1 μM of CAM2(OG) in the absence or presence of 10 μM NBQX, 10 μM AP5, or 10 μM (2S,4R)-4-methyl glutamate (4MG) to examine selective labelling of AMPARs among the ionotropic glutamate receptor family. (c) Analyses of labelled proteins in cultured neurons by immunoprecipitation using anti-Fl/OG antibody. Chemical labelling was conducted with the same procedure described in a. After lysis of labelled cultured neurons by CAM2(Fl), the cell lysate was immunoprecipitated with anti-Fl/OG antibodies. The immunoprecipitates were analyzed by western blot using glutamate receptor-specific antibodies. (d–h) Confocal imaging of cultured neurons after labelling using CAM reagents. Cultured hippocampal neurons labelled with 1 μM CAM2(Fl) were fixed, permeabilized and immunostained using anti-MAP2 (in d,f), anti-GluA2 (in e,g) or anti-PSD95 antibody (in h). White square ROIs indicated in d,e are expanded in f,g, respectively. Scale bars, 10 μm (d,e) and 5 μm (f–h). Full blots for b and c are shown in Supplementary Fig. 22.
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f3: Chemical labelling of native AMPARs in cultured neurons.(a) Western blot analyses of cultured neurons after labelling using CAM reagents. Cultured cortical neurons were treated with 1 μM of CAM2(OG), CAM2(Fl), CAM2(Ax488), or CAM2(Bt) in the absence or presence of 10 μM NBQX in serum free Neurobasal medium. The cell lysates were analyzed by western blot using anti-Fl/OG, anti-Ax488, or anti-GluA2 antibody, or by biotin blotting using streptavidin-HRP. * indicates biotinylated proteins endogenously expressed in the neurons. (b) Effect of competitive antagonists for glutamate receptors on chemical labelling of native AMPARs in cultured neurons. Western blot analyses of cultured neurons after labelling using CAM reagents are shown. Cultured cortical neurons were treated with 1 μM of CAM2(OG) in the absence or presence of 10 μM NBQX, 10 μM AP5, or 10 μM (2S,4R)-4-methyl glutamate (4MG) to examine selective labelling of AMPARs among the ionotropic glutamate receptor family. (c) Analyses of labelled proteins in cultured neurons by immunoprecipitation using anti-Fl/OG antibody. Chemical labelling was conducted with the same procedure described in a. After lysis of labelled cultured neurons by CAM2(Fl), the cell lysate was immunoprecipitated with anti-Fl/OG antibodies. The immunoprecipitates were analyzed by western blot using glutamate receptor-specific antibodies. (d–h) Confocal imaging of cultured neurons after labelling using CAM reagents. Cultured hippocampal neurons labelled with 1 μM CAM2(Fl) were fixed, permeabilized and immunostained using anti-MAP2 (in d,f), anti-GluA2 (in e,g) or anti-PSD95 antibody (in h). White square ROIs indicated in d,e are expanded in f,g, respectively. Scale bars, 10 μm (d,e) and 5 μm (f–h). Full blots for b and c are shown in Supplementary Fig. 22.

Mentions: We next examined whether CAM2 can be applicable to native AMPARs in cultured neurons. Cultured cortical neurons were incubated with CAM2(OG) at 17 °C, and the OG labelling was evaluated using western blot methods. As shown in Fig. 3a, a single protein band corresponding to that of the AMPAR (100–110 kDa) was clearly detected in the presence of CAM2(OG), and this band was absent in the presence of the competitive inhibitor, NBQX. Importantly, this labelling was not blocked in the presence of an NMDA receptor inhibitor (AP5) or a kainite receptor inhibitor ([2S,4R]-4-methyl glutamate (4MG)), implying that the labelling was selective to AMPARs among the ionotropic glutamate receptor family (Fig. 3b). In addition, immunoprecipitation using anti-Fl/OG antibody showed that covalent attachment of the fluorophore was selective for the AMPARs, with none observed for NMDA or kainite receptors (Fig. 3c). Similarly, native neuronal AMPARs were selectively labelled with other CAM2 reagents bearing different probes (CAM2(Fl), CAM2(Ax488) and CAM2(Bt)) (Fig. 3a). Although a lack of probe-specific antibodies precluded western blot analysis for the other probes listed in Fig. 1b, these results indicate that CAM2 could specifically label native AMPARs in cultured neurons.


Chemical labelling for visualizing native AMPA receptors in live neurons
Chemical labelling of native AMPARs in cultured neurons.(a) Western blot analyses of cultured neurons after labelling using CAM reagents. Cultured cortical neurons were treated with 1 μM of CAM2(OG), CAM2(Fl), CAM2(Ax488), or CAM2(Bt) in the absence or presence of 10 μM NBQX in serum free Neurobasal medium. The cell lysates were analyzed by western blot using anti-Fl/OG, anti-Ax488, or anti-GluA2 antibody, or by biotin blotting using streptavidin-HRP. * indicates biotinylated proteins endogenously expressed in the neurons. (b) Effect of competitive antagonists for glutamate receptors on chemical labelling of native AMPARs in cultured neurons. Western blot analyses of cultured neurons after labelling using CAM reagents are shown. Cultured cortical neurons were treated with 1 μM of CAM2(OG) in the absence or presence of 10 μM NBQX, 10 μM AP5, or 10 μM (2S,4R)-4-methyl glutamate (4MG) to examine selective labelling of AMPARs among the ionotropic glutamate receptor family. (c) Analyses of labelled proteins in cultured neurons by immunoprecipitation using anti-Fl/OG antibody. Chemical labelling was conducted with the same procedure described in a. After lysis of labelled cultured neurons by CAM2(Fl), the cell lysate was immunoprecipitated with anti-Fl/OG antibodies. The immunoprecipitates were analyzed by western blot using glutamate receptor-specific antibodies. (d–h) Confocal imaging of cultured neurons after labelling using CAM reagents. Cultured hippocampal neurons labelled with 1 μM CAM2(Fl) were fixed, permeabilized and immunostained using anti-MAP2 (in d,f), anti-GluA2 (in e,g) or anti-PSD95 antibody (in h). White square ROIs indicated in d,e are expanded in f,g, respectively. Scale bars, 10 μm (d,e) and 5 μm (f–h). Full blots for b and c are shown in Supplementary Fig. 22.
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f3: Chemical labelling of native AMPARs in cultured neurons.(a) Western blot analyses of cultured neurons after labelling using CAM reagents. Cultured cortical neurons were treated with 1 μM of CAM2(OG), CAM2(Fl), CAM2(Ax488), or CAM2(Bt) in the absence or presence of 10 μM NBQX in serum free Neurobasal medium. The cell lysates were analyzed by western blot using anti-Fl/OG, anti-Ax488, or anti-GluA2 antibody, or by biotin blotting using streptavidin-HRP. * indicates biotinylated proteins endogenously expressed in the neurons. (b) Effect of competitive antagonists for glutamate receptors on chemical labelling of native AMPARs in cultured neurons. Western blot analyses of cultured neurons after labelling using CAM reagents are shown. Cultured cortical neurons were treated with 1 μM of CAM2(OG) in the absence or presence of 10 μM NBQX, 10 μM AP5, or 10 μM (2S,4R)-4-methyl glutamate (4MG) to examine selective labelling of AMPARs among the ionotropic glutamate receptor family. (c) Analyses of labelled proteins in cultured neurons by immunoprecipitation using anti-Fl/OG antibody. Chemical labelling was conducted with the same procedure described in a. After lysis of labelled cultured neurons by CAM2(Fl), the cell lysate was immunoprecipitated with anti-Fl/OG antibodies. The immunoprecipitates were analyzed by western blot using glutamate receptor-specific antibodies. (d–h) Confocal imaging of cultured neurons after labelling using CAM reagents. Cultured hippocampal neurons labelled with 1 μM CAM2(Fl) were fixed, permeabilized and immunostained using anti-MAP2 (in d,f), anti-GluA2 (in e,g) or anti-PSD95 antibody (in h). White square ROIs indicated in d,e are expanded in f,g, respectively. Scale bars, 10 μm (d,e) and 5 μm (f–h). Full blots for b and c are shown in Supplementary Fig. 22.
Mentions: We next examined whether CAM2 can be applicable to native AMPARs in cultured neurons. Cultured cortical neurons were incubated with CAM2(OG) at 17 °C, and the OG labelling was evaluated using western blot methods. As shown in Fig. 3a, a single protein band corresponding to that of the AMPAR (100–110 kDa) was clearly detected in the presence of CAM2(OG), and this band was absent in the presence of the competitive inhibitor, NBQX. Importantly, this labelling was not blocked in the presence of an NMDA receptor inhibitor (AP5) or a kainite receptor inhibitor ([2S,4R]-4-methyl glutamate (4MG)), implying that the labelling was selective to AMPARs among the ionotropic glutamate receptor family (Fig. 3b). In addition, immunoprecipitation using anti-Fl/OG antibody showed that covalent attachment of the fluorophore was selective for the AMPARs, with none observed for NMDA or kainite receptors (Fig. 3c). Similarly, native neuronal AMPARs were selectively labelled with other CAM2 reagents bearing different probes (CAM2(Fl), CAM2(Ax488) and CAM2(Bt)) (Fig. 3a). Although a lack of probe-specific antibodies precluded western blot analysis for the other probes listed in Fig. 1b, these results indicate that CAM2 could specifically label native AMPARs in cultured 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