Visualization of synaptic Ca2+ /calmodulin-dependent protein kinase II activity in living neurons - PubMed (original) (raw)

Detection of activation of synaptic Camuiα in hippocampal neurons. A, An image of a neuron expressing Camuiα. B, Time-lapse FRET images (intensity-modulated display mode) of the same neuron, stimulated with glutamate (20 μ

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) between time 0 and 5 min. Examples of spontaneous change are indicated by arrowheads. C, Ensemble change in FRET level by glutamate application. FRET level in dendrites is also shown. There were 43 spines from two dendrites, one of which is shown in A and B. D, High-magnification images of the boxed area in A before and after stimulation. Graphs show background-subtracted CFP and YFP fluorescence profiles along a line (width, 5 pixels) across three spines. The background was 289.6 and 294.9 for CFP and 324.2 and 329.0 for YFP before and after the stimulation, respectively. A.U., Arbitrary units. E, F, Plots of unstimulated FRET level and glutamate-induced change versus unstimulated CFP intensity in individual spines. CFP intensity and unstimulated FRET level are the average of three images taken between -10 and 0 min. Dend., Dendrite. G, Anti-phospho-T286 CaMKII and anti-CaMKIIα immunoblots of neurons similarly stimulated. Both Camuiα and endogenous CaMKIIα are shown. Experiments were done in duplicate. Glu, Glutamate; Wash, washout. H, Effect of selective activation of NMDA receptor by NMDA (25 μ

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) and glycine (1 μ

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) (26 spines from 2 neurons).

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-AP-5 (100 μ

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) blocked the response. I, Comparison of the NMDA receptor-mediated response of Camuiα wild-type (WT) and T286A mutant (60 spines from 4 neurons). J, Effect of selective AMPA receptor activation with AMPA (40 μ

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) and blockade of effect with NBQX (1 μ

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; 28 spines from 2 neurons). H-J, Experiments were performed in the presence of TTX (1 μ

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) to prevent secondary release of transmitters. Scale bars, 2 μm. Error bars represent SEM.