Figure 2. KCl-induced localization of ZBP1 granules in dendrites is dependent on NMDA receptor activity.

A, Triple-label fluorescence detection of ZBP1 (red), synaptophysin (blue), and phalloidin (green). The colored boxes overlaid on top of the dendrites show schematically how the dendrite was divided into three regions of interest (proximal, middle, and distal) for performing quantitative digital imaging analysis of pixel intensities with IPLab software (Scanalytics). These three regions have been enlarged at the bottom of each panel by using the same color-coded border. All images were acquired with shorter exposure times than those used in Figure 1, such that the ZBP1 signal in dendrites was sparse and undetectable in distal regions of unstimulated neurons.

B, The addition of 20 mM KCl to the culture medium for 15 min increased ZBP1 levels in all three dendritic regions as compared with unstimulated neurons. After KCl treatment ZBP1 fluorescence was now apparent in distal regions.

C, Histogram of mean ZBP1 immunofluorescence intensities for each treatment; 45 dendrites, one per neuron, were analyzed from three experiments. KCl elicited a statistically significant increase in ZBP1 levels (mean 19.8% increase) as compared with nonstimulated control neurons. The NMDA receptor antagonist AP-5 completely inhibited the KCl response. Exposure of neurons to AP-5 alone for 15 min decreased ZBP levels (mean 12.6% decrease), which was not increased by KCl treatment in the presence of AP-5. Bars show group mean fluorescence intensity/area ± SEM ;*p < 0.01, two-tailed Mann-Whitney t test. Black asterisks denote significance as compared with control untreated neurons. Red asterisks denote significant difference between AP-5+KCl as compared with KCl treatment.