Importantly, GluN1-NMDAR trajectories overlapping the GFP signal of transfected neurons presented a wide range of behaviors, both in cultured and acute slices, ranging from immobile to slowly and highly mobile along dendrites and spines [Figs. 5(a) and 5(b)]. As previously reported in cultured neurons,15 single NMDAR were also found to rapidly alternate between slow and fast diffusion phases in cultured and acute slices [Figs. 5(c) and 5(d)], likely reflecting changing cellular environments explored by the receptor. We calculated the instantaneous diffusion coefficients of each trajectory as described in Sec. 2. When comparing the diffusion coefficients of GluN1-NMDAR in the three systems, it emerged that receptor surface diffusion was slower in acute slices when compared with cultured preparations [Figs. 5(e) and 5(f)]. Noteworthy, the fractions of immobile receptors, defined as presenting diffusion coefficients lower than [first point of the cumulative distribution; Fig. 5(e)], were similar for the three cases. The reduced diffusion in more intact preparations appear thus to be related to a relative increase in slowly mobile NMDAR. We tested whether this apparent reduction in NMDAR mobility results from an increased internalization of receptors. For this, cultured slices were exposed to dynasore () to block dynamin-dependent internalization pathways (including clathrin- and caveolin-ones). We report that NMDAR diffusion was unchanged by the treatment (median instantaneous diffusion : control, 0.01 to , trajectories; dynasore, 0.007 to , trajectories; ), suggesting that the internalization rate/process does not directly contribute to the observed diffusion parameters. The difference in mobility between preparations could however be a consequence of tracking receptors with two-dimensional (2-D) resolution in a three-dimensional (3-D) environment. Indeed, we observed a significant decrease in trajectory length when comparing 2-D dissociated cultures with 3-D cultured organotypic and acute brain slices (mean trajectory : cultures, , trajectories; organotypic slices, , trajectories; acute slices, , trajectories; ***), suggesting that receptors cannot be tracked over long periods of time in brain tissue [Fig. 5(f)]. This is likely due to the fact that mobile receptor-QDs complexes go rapidly out of focus in a 3-D environment and are thus potentially underestimated, favoring the collection of slowly moving receptors. To note, the diffusion of GluN1-NMDAR in both acute and cultured slices was found to be slower than the diffusion of CFP-tagged recombinant dopamine D1 receptors tracked in acute brain slices in identical experimental conditions, in accordance with previous reports showing that D1 receptors are more diffusive than NMDAR in dissociated neuronal cultures.32 To further test whether the pattern of diffusing GluN1-NMDAR was different between cultured and acute brain slices, we randomly selected a subset of long GluN1-NMDAR trajectories ( frames duration) in each condition and calculated their respective MSD. As shown in Fig. 6, initial phases of the MSD versus time plots were not different when comparing cultured and acute brain slices, suggesting that the pattern of GluN1-NMDAR surface exploration was relatively similar at short lag times in both preparations. However, the surface explored by receptors appeared to be significantly larger in cultured organotypic slices at longer lag times, indicating a higher level of receptor confinement in acute preparations [Fig. 6(b)].