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LIS1-dependent retrograde translocation of excitatory synapses in developing interneuron dendrites.

Kawabata I, Kashiwagi Y, Obashi K, Ohkura M, Nakai J, Wynshaw-Boris A, Yanagawa Y, Okabe S - Nat Commun (2012)

Bottom Line: This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility.Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites.Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Synaptic remodelling coordinated with dendritic growth is essential for proper development of neural connections. After establishment of synaptic contacts, synaptic junctions are thought to become stationary and provide fixed anchoring points for further dendritic growth. However, the possibility of active translocation of synapses along dendritic protrusions, to guide the proper arrangement of synaptic distribution, has not yet been fully investigated. Here we show that immature dendrites of γ-aminobutyric acid-positive interneurons form long protrusions and that these protrusions serve as conduits for retrograde translocation of synaptic contacts to the parental dendrites. This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility. Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites. Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

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Translocation of synaptic contact sites regulated by neuronal activity.(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.
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f4: Translocation of synaptic contact sites regulated by neuronal activity.(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.

Mentions: In dissociated cultures, 85–91% of PSD-95-GFP puncta were associated with presynaptic markers (Supplementary Fig. S2). In cortical slices, 78–80% of PSD-95-GFP clusters in dendritic protrusions were also associated with presynaptic markers. Furthermore, a large fraction of PSD-95-positive protrusions (88–93%) were in contact with presynaptic markers (synapsin I, synaptophysin) in two types of cultures. These results indicate that the majority of imaged PSD puncta were already associated with presynaptic axons. To confirm the simultaneous movement of presynaptic and postsynaptic components, we also recorded time-lapse images of PSD-95-YFP puncta in postsynaptic neurons together with synaptophysin-CFP puncta in axons that are in contact (Fig. 4a). These two fluorescent probes were expressed in only a subset of neurons in dissociated culture by recombinant adenovirus-mediated transfection6; therefore, only a fraction of synaptic junctions showed co-localization of these presynaptic and postsynaptic markers. The movement of PSD-95-YFP and synaptophysin-CFP puncta was highly coordinated (Fig. 4b). The fraction of mobile pairs of PSD-95-YFP puncta and synaptophysin-CPF puncta was comparable with the total mobile fraction of PSD-95-YFP puncta and that of synaptophysin-CFP puncta (Fig. 4c). This result excludes the possibility that PSD-95 clusters with synaptic contacts were less motile in comparison with clusters without contacts. To confirm the presence of functional presynaptic structures, we labelled active presynaptic sites by FM1-43. The movement of PSD-95-TagRFP was highly correlated with that of FM1-43 puncta (Fig. 4d and e), and the average velocity of FM1-43 puncta was comparable to that of PSD-95-TagRFP (Fig. 4f).


LIS1-dependent retrograde translocation of excitatory synapses in developing interneuron dendrites.

Kawabata I, Kashiwagi Y, Obashi K, Ohkura M, Nakai J, Wynshaw-Boris A, Yanagawa Y, Okabe S - Nat Commun (2012)

Translocation of synaptic contact sites regulated by neuronal activity.(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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f4: Translocation of synaptic contact sites regulated by neuronal activity.(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.
Mentions: In dissociated cultures, 85–91% of PSD-95-GFP puncta were associated with presynaptic markers (Supplementary Fig. S2). In cortical slices, 78–80% of PSD-95-GFP clusters in dendritic protrusions were also associated with presynaptic markers. Furthermore, a large fraction of PSD-95-positive protrusions (88–93%) were in contact with presynaptic markers (synapsin I, synaptophysin) in two types of cultures. These results indicate that the majority of imaged PSD puncta were already associated with presynaptic axons. To confirm the simultaneous movement of presynaptic and postsynaptic components, we also recorded time-lapse images of PSD-95-YFP puncta in postsynaptic neurons together with synaptophysin-CFP puncta in axons that are in contact (Fig. 4a). These two fluorescent probes were expressed in only a subset of neurons in dissociated culture by recombinant adenovirus-mediated transfection6; therefore, only a fraction of synaptic junctions showed co-localization of these presynaptic and postsynaptic markers. The movement of PSD-95-YFP and synaptophysin-CFP puncta was highly coordinated (Fig. 4b). The fraction of mobile pairs of PSD-95-YFP puncta and synaptophysin-CPF puncta was comparable with the total mobile fraction of PSD-95-YFP puncta and that of synaptophysin-CFP puncta (Fig. 4c). This result excludes the possibility that PSD-95 clusters with synaptic contacts were less motile in comparison with clusters without contacts. To confirm the presence of functional presynaptic structures, we labelled active presynaptic sites by FM1-43. The movement of PSD-95-TagRFP was highly correlated with that of FM1-43 puncta (Fig. 4d and e), and the average velocity of FM1-43 puncta was comparable to that of PSD-95-TagRFP (Fig. 4f).

Bottom Line: This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility.Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites.Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Synaptic remodelling coordinated with dendritic growth is essential for proper development of neural connections. After establishment of synaptic contacts, synaptic junctions are thought to become stationary and provide fixed anchoring points for further dendritic growth. However, the possibility of active translocation of synapses along dendritic protrusions, to guide the proper arrangement of synaptic distribution, has not yet been fully investigated. Here we show that immature dendrites of γ-aminobutyric acid-positive interneurons form long protrusions and that these protrusions serve as conduits for retrograde translocation of synaptic contacts to the parental dendrites. This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility. Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites. Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

Show MeSH
Related in: MedlinePlus