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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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Effect of manipulating the Lis1 gene in cortical slices.(a) Time-lapse images of an interneuron maintained in cortical slices from a Lis1hc/+ 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 Lis1hc/+ 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.
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f7: Effect of manipulating the Lis1 gene in cortical slices.(a) Time-lapse images of an interneuron maintained in cortical slices from a Lis1hc/+ 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 Lis1hc/+ 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.

Mentions: To confirm the involvement of LIS1 in PSD mobility, we used an alternative genetic approach. The function of LIS1 was decreased by Cre-dependent excision of the Lis1 allele in slice cultures. Mutant mice heterozygous for a conditional knockout/hypomorphic allele (Lis1hc/+) showed normal cortical development. After Cre-dependent excision of a single Lis1 allele, however, the resulting heterozygous mutants (Lis1−/+) exhibited clear structural disorganization of the cortex25. To visualize PSD puncta in Cre-active neurons, expression plasmids for Cre and PSD-95-GFP were introduced into cortical slice preparations by electroporation. The activity of Cre recombinase was confirmed by including an indicator plasmid that expresses RFP only after removal of a loxP-transcription stopper-loxP sequence by Cre recombinase activity (Supplementary Fig. S3). To reveal synapse remodelling in Lis1−/+ interneurons, we performed time-lapse imaging experiments at 4–9 DIV. The overall density and dynamics of dendritic protrusions were indistinguishable between Lis1−/+ and wild-type neurons (density: 0.17±0.015 per μm in wild type and 0.19±0.030 per μm in Lis1−/+, lifetime: 19.6±3.9 min in wild type and 21.1±5.6 min in Lis1−/+; n=12 cells for wild type and n=9 cells for Lis1−/+). However, the fraction of PSD puncta localized in the protrusions was reduced in Lis1−/+ neurons (18.8±3.5% in wild type and 6.7±2.4% in Lis1−/+, n=12 and 9 cells). Furthermore, when PSD puncta were present within protrusions, their mobility was significantly suppressed (Fig. 7a,b).


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)

Effect of manipulating the Lis1 gene in cortical slices.(a) Time-lapse images of an interneuron maintained in cortical slices from a Lis1hc/+ 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 Lis1hc/+ 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.
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f7: Effect of manipulating the Lis1 gene in cortical slices.(a) Time-lapse images of an interneuron maintained in cortical slices from a Lis1hc/+ 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 Lis1hc/+ 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.
Mentions: To confirm the involvement of LIS1 in PSD mobility, we used an alternative genetic approach. The function of LIS1 was decreased by Cre-dependent excision of the Lis1 allele in slice cultures. Mutant mice heterozygous for a conditional knockout/hypomorphic allele (Lis1hc/+) showed normal cortical development. After Cre-dependent excision of a single Lis1 allele, however, the resulting heterozygous mutants (Lis1−/+) exhibited clear structural disorganization of the cortex25. To visualize PSD puncta in Cre-active neurons, expression plasmids for Cre and PSD-95-GFP were introduced into cortical slice preparations by electroporation. The activity of Cre recombinase was confirmed by including an indicator plasmid that expresses RFP only after removal of a loxP-transcription stopper-loxP sequence by Cre recombinase activity (Supplementary Fig. S3). To reveal synapse remodelling in Lis1−/+ interneurons, we performed time-lapse imaging experiments at 4–9 DIV. The overall density and dynamics of dendritic protrusions were indistinguishable between Lis1−/+ and wild-type neurons (density: 0.17±0.015 per μm in wild type and 0.19±0.030 per μm in Lis1−/+, lifetime: 19.6±3.9 min in wild type and 21.1±5.6 min in Lis1−/+; n=12 cells for wild type and n=9 cells for Lis1−/+). However, the fraction of PSD puncta localized in the protrusions was reduced in Lis1−/+ neurons (18.8±3.5% in wild type and 6.7±2.4% in Lis1−/+, n=12 and 9 cells). Furthermore, when PSD puncta were present within protrusions, their mobility was significantly suppressed (Fig. 7a,b).

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