Huang YB et al. (2015), In Vivo Study of Dynamics and Stability of Dend...

XB-ART-51440

PLoS One 2015 Oct 20;1010:e0140752. doi: 10.1371/journal.pone.0140752.

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In Vivo Study of Dynamics and Stability of Dendritic Spines on Olfactory Bulb Interneurons in Xenopus laevis Tadpoles.

Huang YB , Hu CR , Zhang L , Yin W , Hu B .

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Dendritic spines undergo continuous remodeling during development of the nervous system. Their stability is essential for maintaining a functional neuronal circuit. Spine dynamics and stability of cortical excitatory pyramidal neurons have been explored extensively in mammalian animal models. However, little is known about spiny interneurons in non-mammalian vertebrate models. In the present study, neuronal morphology was visualized by single-cell electroporation. Spiny neurons were surveyed in the Xenopus tadpole brain and observed to be widely distributed in the olfactory bulb and telencephalon. DsRed- or PSD95-GFP-expressing spiny interneurons in the olfactory bulb were selected for in vivo time-lapse imaging. Dendritic protrusions were classified as filopodia, thin, stubby, or mushroom spines based on morphology. Dendritic spines on the interneurons were highly dynamic, especially the filopodia and thin spines. The stubby and mushroom spines were relatively more stable, although their stability significantly decreased with longer observation intervals. The 4 spine types exhibited diverse preferences during morphological transitions from one spine type to others. Sensory deprivation induced by severing the olfactory nerve to block the input of mitral/tufted cells had no significant effects on interneuron spine stability. Hence, a new model was established in Xenopus laevis tadpoles to explore dendritic spine dynamics in vivo.

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Species referenced: Xenopus laevis
Genes referenced: dlg4

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	Fig 2. In vivo images of DsRed2 and PSD95-GFP double-labeled spiny neurons in X. laevis tadpoles.(A) A spiny neuron was labeled by DsRed2 and PSD95-GFP. The postsynaptic specializations on spines are shown as yellow puncta. Green dots are developing melanocyte pigmentation not fully inhibited by phenylthiocarbamide (PTU). (B, C) High magnification views showing the different types of dendritic spines of the spiny neuron in (A). F: filopodia; T: thin spine; S: stubby spine; M: mushroom spine. The yellow puncta represent synaptic contacts, most of which were on stubby and mushroom spines. (D) Statistical analysis of the percentage of PSD95-GFP on different types of dendritic spines. Stubby and mushroom spines exhibited a higher number of PSD95-GFP puncta than filopodia and thin spines. Bars indicate mean ± SEM. N = 4 neurons with 9 dendrites were used for colocalization analysis of PSD95-GFP. The significance levels wereP < 0.05, P < 0.01, and **P < 0.001.
	Fig 3. Stability and dynamics of dendritic spines during short-term observation.(A) Serial time-lapse images of a single neuron showing stable spines (filled arrowheads) and dynamic spines (open arrowheads) at 15-min intervals. (B) Stability of the 4 types of dendritic spines was observed at 15-min intervals. The stubby and mushroom spines were more stable than the filopodia and thin spines. (C) Detailed analysis of the stability of filopodia and thin spines versus stubby and mushroom spines at each 15-min interval observation. (D, E) Detailed analysis of filopodia and thin spines versus stubby and mushroom spines that were added (D) or eliminated (E) during each 15-min interval observation. (F) Average dendritic spine stability and dynamics at each 15-min observation. Bars indicate means ± SEM. N = 4 neurons with 9 dendrites were used for the 15-min short-term observation. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 4. Stability and dynamics of dendritic spines during long-term observation.(A) Serial time-lapse images of a dendritic branch showing stable spines (filled arrowheads) and dynamic spines (open arrowheads) at 2-h intervals. (B) Stability of the 4 types of dendritic spines was observed at 2-h intervals. (C) Detailed analysis of stability of filopodia and thin spines versus stubby and mushroom spines during each 2-h interval observation. (D, E) Detailed analysis of filopodia and thin spines versus stubby and mushroom spines that were added (D) or eliminated (E) during each 2-h interval observation. (F) Average data of dendritic spine stability and dynamics at each 2-h observation. (G) Stability of the dendritic spines at different time interval observations. Bars indicate means ± SEM. N = 4 neurons with 7 dendrites were used for the 2-h and 24-h long-term observations. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 5. Transformation of dendritic protrusions into other spine types.(A) A mushroom spine (indicated by an arrow in A1) transformed into a thin spine at 30 min (A3), and reverted to a mushroom spine at 60 min (A5). Another thin spine (indicated by an arrowhead) transformed into a mushroom spine at 30 min (A3). (B) A stubby spine (indicated by an arrow in B1) transformed into a mushroom spine at 2 h (B2), remained in that shape up to 6 h (B4), and became a thin spine at 24 h (B5). “+” indicates newly added spines, and “-” indicates eliminated spines. (C) The percentage of spines that transformed into other forms during the pooled data of 15-min and 2-h interval observations. (D–G) Percentage of each spine type that transformed into other types. (D) All filopodia transformed into thin spines, and a few transformed into large spines (P < 0.05). (E) Some changed thin spines transformed into filopodia, and some transformed into large spines; no significant difference was observed between these two forms. (F) Most of the stubby spines transformed into mushroom spines, and only a few transformed into small spines (P < 0.001). (G) Most of the mushroom spines transformed into stubby spines, and only a few transformed into small spines (P < 0.05). Figure labels: trans. = transformation, fil. = filopodia, stu. = stubby, and mus. = mushroom. Neurons used for transformation analysis were from the 15-min short-term and 2-h middle-term observations. Bars indicate means ± SEM. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 6. Stability of dendritic spines after olfactory nerve severance.(A, B) Serial time-lapse images showing the morphology of dendritic spines in the sham group (A) and olfactory nerve severance group (B) imaged at 0 h, 4 h, and 24 h, respectively, after surgery. (C, D) Stability of small and large spines after sensory deprivation compared with normal spines during the 2-h and 24-h observation periods. (E) Density of the spines after surgery. Bars indicate means ± SEM. N = 10 neurons with 16 dendrites were used for the sensory deprivation analysis. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 1. Distribution of spiny neurons in the Xenopus tadpole brain.Fluorescent dye was electroporated into neurons located in different brain regions. (A1, A2) Spiny neuron in the OB. (A1) Electrode filled with fluorescent dye inserted into the OB, pointing to a labeled neuron. (A2) High magnification of the neuron in A1. Inset shows spines on a dendrite. (B1, B2) Mitral cell in the anterior OB. (C1, C2) Spiny neuron in the telencephalon (Tel). (D1, D2) Non-spiny neuron in the tectum (Tec). (E) Diagram summarizing all labeled spiny (red) and non-spiny (blue) neurons in different brain areas in the left hemisphere. Twenty neurons randomly labeled in the optic tectum were non-spiny neurons; 69 neurons in the OB and telencephalon were spiny neurons except for 11 neurons.

References [+] :

Alsina, Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF. 2001, Pubmed, Xenbase

	Fig 2. In vivo images of DsRed2 and PSD95-GFP double-labeled spiny neurons in X. laevis tadpoles.(A) A spiny neuron was labeled by DsRed2 and PSD95-GFP. The postsynaptic specializations on spines are shown as yellow puncta. Green dots are developing melanocyte pigmentation not fully inhibited by phenylthiocarbamide (PTU). (B, C) High magnification views showing the different types of dendritic spines of the spiny neuron in (A). F: filopodia; T: thin spine; S: stubby spine; M: mushroom spine. The yellow puncta represent synaptic contacts, most of which were on stubby and mushroom spines. (D) Statistical analysis of the percentage of PSD95-GFP on different types of dendritic spines. Stubby and mushroom spines exhibited a higher number of PSD95-GFP puncta than filopodia and thin spines. Bars indicate mean ± SEM. N = 4 neurons with 9 dendrites were used for colocalization analysis of PSD95-GFP. The significance levels wereP < 0.05, P < 0.01, and **P < 0.001.
	Fig 3. Stability and dynamics of dendritic spines during short-term observation.(A) Serial time-lapse images of a single neuron showing stable spines (filled arrowheads) and dynamic spines (open arrowheads) at 15-min intervals. (B) Stability of the 4 types of dendritic spines was observed at 15-min intervals. The stubby and mushroom spines were more stable than the filopodia and thin spines. (C) Detailed analysis of the stability of filopodia and thin spines versus stubby and mushroom spines at each 15-min interval observation. (D, E) Detailed analysis of filopodia and thin spines versus stubby and mushroom spines that were added (D) or eliminated (E) during each 15-min interval observation. (F) Average dendritic spine stability and dynamics at each 15-min observation. Bars indicate means ± SEM. N = 4 neurons with 9 dendrites were used for the 15-min short-term observation. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 4. Stability and dynamics of dendritic spines during long-term observation.(A) Serial time-lapse images of a dendritic branch showing stable spines (filled arrowheads) and dynamic spines (open arrowheads) at 2-h intervals. (B) Stability of the 4 types of dendritic spines was observed at 2-h intervals. (C) Detailed analysis of stability of filopodia and thin spines versus stubby and mushroom spines during each 2-h interval observation. (D, E) Detailed analysis of filopodia and thin spines versus stubby and mushroom spines that were added (D) or eliminated (E) during each 2-h interval observation. (F) Average data of dendritic spine stability and dynamics at each 2-h observation. (G) Stability of the dendritic spines at different time interval observations. Bars indicate means ± SEM. N = 4 neurons with 7 dendrites were used for the 2-h and 24-h long-term observations. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 5. Transformation of dendritic protrusions into other spine types.(A) A mushroom spine (indicated by an arrow in A1) transformed into a thin spine at 30 min (A3), and reverted to a mushroom spine at 60 min (A5). Another thin spine (indicated by an arrowhead) transformed into a mushroom spine at 30 min (A3). (B) A stubby spine (indicated by an arrow in B1) transformed into a mushroom spine at 2 h (B2), remained in that shape up to 6 h (B4), and became a thin spine at 24 h (B5). “+” indicates newly added spines, and “-” indicates eliminated spines. (C) The percentage of spines that transformed into other forms during the pooled data of 15-min and 2-h interval observations. (D–G) Percentage of each spine type that transformed into other types. (D) All filopodia transformed into thin spines, and a few transformed into large spines (P < 0.05). (E) Some changed thin spines transformed into filopodia, and some transformed into large spines; no significant difference was observed between these two forms. (F) Most of the stubby spines transformed into mushroom spines, and only a few transformed into small spines (P < 0.001). (G) Most of the mushroom spines transformed into stubby spines, and only a few transformed into small spines (P < 0.05). Figure labels: trans. = transformation, fil. = filopodia, stu. = stubby, and mus. = mushroom. Neurons used for transformation analysis were from the 15-min short-term and 2-h middle-term observations. Bars indicate means ± SEM. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 6. Stability of dendritic spines after olfactory nerve severance.(A, B) Serial time-lapse images showing the morphology of dendritic spines in the sham group (A) and olfactory nerve severance group (B) imaged at 0 h, 4 h, and 24 h, respectively, after surgery. (C, D) Stability of small and large spines after sensory deprivation compared with normal spines during the 2-h and 24-h observation periods. (E) Density of the spines after surgery. Bars indicate means ± SEM. N = 10 neurons with 16 dendrites were used for the sensory deprivation analysis. Significance was set at P < 0.05, P < 0.01, and **P < 0.001.
	Fig 1. Distribution of spiny neurons in the Xenopus tadpole brain.Fluorescent dye was electroporated into neurons located in different brain regions. (A1, A2) Spiny neuron in the OB. (A1) Electrode filled with fluorescent dye inserted into the OB, pointing to a labeled neuron. (A2) High magnification of the neuron in A1. Inset shows spines on a dendrite. (B1, B2) Mitral cell in the anterior OB. (C1, C2) Spiny neuron in the telencephalon (Tel). (D1, D2) Non-spiny neuron in the tectum (Tec). (E) Diagram summarizing all labeled spiny (red) and non-spiny (blue) neurons in different brain areas in the left hemisphere. Twenty neurons randomly labeled in the optic tectum were non-spiny neurons; 69 neurons in the OB and telencephalon were spiny neurons except for 11 neurons.