(a) Upper right panel: a hippocampal-dissociated neuron expressing PSD-95-GFP with the typical morphology of an interneuron. The GABAergic phenotype of this neuron was confirmed by anti-GAD67 immunostaining. Upper left panel: a GAD67-negative neuron extending branched dendrites in a dissociated culture. This neuron exhibits the typical morphology of hippocampal pyramidal neurons in culture. Lower right panel: RFP expression in layer II–III non-pyramidal neurons in cortical slices prepared from GAD67-GFP knockin mice. The RFP-positive non-pyramidal neuron (arrows) was also GFP-positive, indicating its GABAergic transmitter phenotype. Lower left panel: RFP-expressing pyramidal-shaped neuron (arrowheads) was GFP-negative. Scale bars, 50 μm. (b) Distribution of PSD-95-GFP puncta and dendritic protrusions in non-pyramidal neurons in a dissociated hippocampal culture at 12 and 19 DIV and a cortical slice culture at 7 and 14 DIV. Dendritic protrusions (arrows) were specifically enriched in immature dendrites and were positive for PSD-95-GFP puncta. Scale bar, 10 μm. (c) Distribution of protrusion lengths in cortical slices at 7 and 14 DIV. At both time points, 2–4 dendritic segments of 12 neurons from 4 slices were imaged and the same number of dendritic protrusions was randomly selected for quantification. (d) The mean lengths of dendritic protrusions at 7 and 14 DIV with or without PSD-95-GFP puncta (PSD+ or PSD−; n=12 neurons from 4 slices at both time points). Significant differences (one-way analysis of variance followed by Tukey–Kramer multiple comparison tests): *P<0.05; **P<0.01. All numeric data are mean±s.e.m.

(a) Time-lapse images of protrusions in pyramidal neurons and interneurons expressing RFP at 9–12 DIV in dissociated cultures. Confocal image stacks were obtained every 10 min, and maximum-intensity projection images were generated. Arrows indicate typical dendritic protrusions on interneurons. Scale bar, 10 μm. (b) Time-lapse images of protrusive activity in pyramidal neurons and interneurons expressing RFP at 6–9 DIV in slices. Before image acquisition, the identities of the neurons were confirmed by GAD67-GFP fluorescence. Confocal image stacks were obtained every 3 min, and maximum-intensity projection images were generated. Scale bar, 5 μm. (c,d) Cumulative frequency plots of dendritic protrusion length and lifetime. Dendritic protrusions in interneurons were longer and more stable than those in pyramidal neurons. From the images of one to three dendritic segments of a single cell, five protrusions were randomly selected. For both interneurons and pyramidal neurons, data from 15 neurons were pooled and compared. (e) Two-dimensional plots of protrusion lifetime and length. Stable interneuron protrusions tend to be longer. (f) Overlay of two-dimensional plots of protrusion lifetime and length. The extensive overlap of data points suggests that the differences in average lengths and lifetimes do not reflect the existence of distinct classes of protrusions in interneurons.

(a) Time-lapse images of an interneuron dendrite expressing PSD-95-GFP and RFP in a dissociated hippocampal culture. A PSD-95-GFP punctum (arrows) moved in a distal to proximal direction towards the dendritic shaft. Scale bar, 3 μm. (b) Time-lapse images of an interneuron dendrite expressing GFP-Homer1c and RFP in a cortical slice culture. A GFP-Homer1c punctum (arrows) moved in a distal to proximal direction towards a stationary punctum at the base of a protrusion. Scale bar, 5 μm. (c,d) Distinct behaviours of mobile and stationary PSD-95-GFP puncta. Mobile PSD puncta showed unidirectional movement of >3 μm in distance (shaded area). Stationary PSD puncta showed limited lateral movement along dendritic protrusion with no preferred direction. Scale bars, 5 μm. (e) The mobile fraction and translocation distance of PSD-95 clusters, but not translocation speed, were lower in dissociated hippocampal interneurons at 18–21 DIV than at 9–12 DIV. (n=16 cells at 9–12 DIV, n=8 cells at 18–21 DIV. The number of PSD-95-positive protrusions/PSD-95 puncta analysed per cell was 2–8 protrusions/2–10 puncta at 9–12 DIV and 2–8 protrusions/3–12 puncta at 18–21 DIV.) Significant differences (unpaired Student's t-test): *P<0.05; **P<0.01. (f) The mobile fraction of PSD-95 clusters in cortical slice preparations was lower at 13–18 DIV than at 4–9 DIV. Translocation distance of PSD-95 clusters was lower in interneurons at 13–18 DIV compared with 4–9 DIV, but the difference was not statistically significant. The speed of translocation was comparable at both developmental stages. (n=16 cells from 16 slice cultures at DIV 4–9, n=5 cells from 5 slice cultures at DIV 13–18. The number of PSD-95-positive protrusions/PSD-95 puncta analysed per cell was 1–5 protrusions/2–9 puncta at DIV 4–9 and 3–8 protrusions/4–14 puncta at DIV 13–18.) Significant differences (unpaired Student's t-test): *P<0.05. All numeric data are mean±s.e.m.

(a–c) Coordinated movement of a presynaptic bouton detected by synaptophysin-CFP fluorescence and a postsynaptic PSD-95-YFP punctum (arrows in a). The graph in b indicates the coordinated change in the position of the presynaptic and postsynaptic components. The fraction of mobile pairs of PSD-95-YFP and synaptophysin-CPF (29±6%, n=7 cells) was comparable with the total mobile fraction of PSD-95-YFP (28±3%, n=7 cells) and synaptophysin-CFP (27±6%, n=7 cells) (c). (d–f) Coordinated movement of an FM1-43 punctum and a PSD-95-TagRFP punctum (arrows in d). The graph in e indicates the coordinated change in the position of two fluorescent puncta. The averaged positions of mobile FM1-43 and PSD-95-TagRFP puncta (f) are also highly coordinated with similar velocities (FM1-43: 2.1±0.4 μm h⁻¹; PSD-95: 2.1±0.5 μm h⁻¹; n=4 cells from four culture preparations). (g–i) NMDA receptor-dependent local calcium transients at the sites of mobile PSD-95 puncta. Translocation of PSD-95-TagRFP puncta along the dendritic protusions (arrowheads in g) was monitored simultaneously with local calcium transients detected by G-CaMP6 (h). G-CaMP6 images correspond to the time point of 5 min in g. The frequency of the local calcium transients was comparable between mobile PSD-95 puncta and stationary PSD-95 puncta in dendritic shafts (i; 0.63±0.20 versus 0.48±0.15 min⁻¹, n=11 puncta from four independent experiments for both protrusions and shafts). Application of APV completely suppressed local calcium transients (no event in four experiments). (j–l) Increased spontaneous firing evoked by bicuculline blocked synapse translocation. Translocation of PSD-95-TagRFP was monitored before and after application of bicuculline (arrowheads in j). Bicuculline treatment induced global calcium transients detected by G-CaMP6 (k), possibly corresponding to bursts of action potentials. Comparison of the puncta velocity revealed a reduction in mobility under bicuculline treatment (l; n=4 cells from four independent experiments for both conditions). Significant differences (paired Student's t-test): **P<0.01. Scale bars, 5 μm for a,d,g, 2 μm for j. All numeric data are mean±s.e.m.

(a) Time-lapse imaging of an EB3-GFP and RFP-expressing neuron showing penetration of an EB3-GFP comet (arrowheads) into a dendritic protrusion. Scale bar, 5 μm. (b) Immunocytochemical staining of interneurons with an anti-MAP2 antibody after time-lapse imaging of PSD-95-GFP. Arrows indicate a PSD-95-GFP punctum moving along a MAP2-positive protrusion. The neuron was fixed 15 min after live imaging. Scale bar, 5 μm. (c–e) Electron micrograph showing microtubules within a dendritic protrusion (double arrows in d and e) of an interneuron. The neuron expressing PSD-95-GFP and RFP was imaged to confirm the presence of PSD-95 puncta in a dendritic protrusion (arrows in c). The same protrusion was identified under an electron microscope to reveal the detail of the cytoplasmic structure. This dendritic protrusion was apposed to a presynaptic bouton filled with synaptic vesicles (asterisk in d). Scale bars, 5 μm for light microscopic images, 200 nm for electron micrographs. (f) Pharmacological manipulation of retrograde PSD mobility. Perturbation of microtubules by vincristine (1 μM) decreased the mobile fraction of PSD-95 puncta, whereas actin perturbation by latrunculin A (5 μM) tended to enhance PSD mobility. Blockade of both voltage-gated sodium channels and ionotropic glutamate receptors by applying a mixture of TTX (1 μM), CNQX (10 μM) and APV (50 μM) increased the mobile fraction of PSDs. Activation of NMDA-type glutamate receptors by D-serine (10 μM) inhibited PSD mobility. (n=5 cells for all the conditions.) Significant differences (paired Student's t-test): *P<0.05. All numeric data are mean±s.e.m.

(a) Fraction of mobile PSD puncta in interneurons transfected with PSD-95-GFP and RFP (control) or PSD-95-GFP, RFP and respective short hairpin RNA (shRNA) constructs. Two additional methods for inhibiting dynein-based mobility were also included (treatment with 1 mM erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) and dynamitin transfection). PSD mobility was suppressed by LIS1 or NDEL1 shRNA and rescued by overexpression of RNAi-insensitive LIS1 and NDEL1 mutants ('LIS1 RNAi+mLIS1' and 'NDEL1 RNAi+mNDEL1'). Mutant LIS1 and NDEL1 RNAi constructs containing three mismatched nucleotides ('mLIS1 RNAi' and 'mNDEL1 RNAi') did not suppress PSD mobility. Both EHNA treatment and expression of dynamitin suppressed PSD mobility. (n=18 cells for control, n=6 cells for LIS1 RNAi, n=7 cells for mLIS1 RNAi, n=6 cells for LIS1 RNAi+mLIS1, n=8 cells for NDEL1 RNAi, n=5 cells for mNDEL1 RNAi, n=5 cells for NDEL1 RNAi+mNDEL1, n=5 cells for EHNA treatment, n=5 cells for dynamitin transfection.) Significant differences (one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests): *P<0.05. (b) Time-lapse imaging of an interneuron expressing PSD-95-GFP with NDEL1-KillerRed before and after CALI (within 60 min after irradiation). Arrowheads indicate the position of a PSD-95-GFP punctum. Scale bar, 5 μm. (c) Suppression of PSD mobility by CALI of NDEL1-KillerRed. The effect of CALI on the mobile fraction was significant at 5–50 min after CALI (53±13.4%, n=6 cells). The fraction of motile PSD-95-GFP puncta returned to the control level at 60–105 min after CALI (91±15.6%, n=6 cells). The mean velocity of mobile PSD puncta also decreased after CALI and this effect was sustained for more than 60 min (2.8±0.30 μm h⁻¹ before CALI, 1.1±0.17 μm h⁻¹ at 5–50 min after CALI, 1.9±0.24 μm h⁻¹ at 60–105 min after CALI, n=6 cells). Significant differences (one-way ANOVA followed by Tukey–Kramer multiple comparison tests): *P<0.05. All numeric data are mean±s.e.m.

(a) Time-lapse images of an interneuron maintained in cortical slices from a Lis1^hc/+ mouse and transfected with expression plasmids encoding Cre recombinase and PSD-95-GFP, together with a Cre indicator plasmid. Arrowheads indicate stationary PSD puncta within dendritic protrusions. Scale bar, 5 μm. (b) Fraction of mobile PSD-95-GFP puncta in dendritic protrusions of control and Lis1^−/+ interneurons (31.2±7.3% for wild type and 7.4±4.9% for Lis1^−/+, n=12 cells in nine slice cultures for wild type, n=9 cells in nine slice cultures for Lis1^−/+). Significant differences (unpaired Student's t-test): *P<0.05. (c) Distribution and morphology of PSD-95-GFP puncta in cortical interneurons from wild-type or Lis1^hc/+ mice and transfected with expression plasmids encoding Cre recombinase and PSD-95-GFP, together with a Cre indicator plasmid. Scale bar, 5 μm. (d) Total fluorescence of individual PSD-95-GFP puncta was lower in interneurons of the Lis1^−/+ genotype than in wild-type interneurons (Lis1^−/+: 140±12 a.u., n=28 cells in seven slice cultures; wild type: 215±25 a.u., n=27 cells from seven slice cultures). Significant differences (unpaired Student's t-test): *P<0.05. (e) Larger variation in the total fluorescence of PSD-95 puncta in interneurons carrying the Lis1^−/+ genotype. The average coefficient of variation for the total fluorescence of PSD-95 puncta in each dendrite was significantly larger in Lis1^−/+ interneurons (Lis1^−/+: 0.66±0.039, n=27 cells from seven slice cultures; wild type: 0.46±0.029, n=28 cells from seven slice cultures). Significant differences (unpaired Student's t-test): **P<0.01. All numeric data are mean±s.e.m.

(a) An interneuron dendrite without protrusions. There is no physical contact with nearby axons. (b) A dendritic protrusion starts contact with a presynaptic axon. Assembly of plus end-distal microtubules in newly formed dendritic protrusions initiate retrograde translocation of PSDs towards the dendritic shafts, while keeping contact with apposed presynaptic boutons. Tight interaction of presynaptic and postsynaptic plasma membranes may transduce the tension generated in the dendritic protrusions to the presynaptic structure. Initiation of PSD mobility may require direct synaptic contacts or diffusible factors released from the axon. (c) Movement of PSDs along dendritic protrusions results in transfer of synapses to the dendritic shaft. (d) On the parental dendritic shafts, where microtubule polarity is mixed, PSD mobility is suppressed and PSD positions are stabilized. It is also possible that activity of the nearby shaft synapses and depolarization of the dendritic shafts contributes to the suppression of PSD mobility.