The role of synaptotagmin I C2A calcium‐binding domain in synaptic vesicle clustering during synapse formation

Gardzinski, Peter; Lee, David W. K.; Zhang, Xu; Hui, Kwokyin; Huang, Guan Jhong; Sun, Hong‐Shuo; Feng, Zhong‐Ping

doi:10.1113/jphysiol.2006.127472

Cited by 23 publications

(20 citation statements)

References 55 publications

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“…This method enables the expression of the GFP reporter in the presynaptic vesicles of dA1 axonal termini. The cerebellum was sectioned at E13.5 and stained with the general pre-synaptic marker synaptotagmin 18,19 , as well as with calbindin that labels the Purkinje layer (Figures 3A, 3B). SV2-GFP + synaptic vesicles were detected in multiple cerebellar regions, including the Purkinje layer, indicating the synaptic connections of dA1 hindbrain interneurons in the cerebellum.…”

Section: Representative Resultsmentioning

confidence: 99%

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Kohl

Hadas

Klar

et al. 2013

JoVE

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Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Loxbased plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development. Video LinkThe video component of this article can be found at

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Section: Representative Resultsmentioning

confidence: 99%

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Kohl

Hadas

Klar

et al. 2013

JoVE

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“…Freshwater snails, L. stagnalis (a hermaphrodite), were kept in water at 20°C under a 12 h light/dark cycle and fed green leaf lettuce twice a week (Gardzinski et al, 2007;Hui et al, 2007). Adult snails with shell lengths of 15-20 mm were used.…”

Section: Methodsmentioning

confidence: 99%

“…Confocal images were acquired using a TCS SL laser confocal microscope (Leica confocal software, version 2.5; build 1347; Leica Microsystems), as described previously (Gardzinski et al, 2007;Hui et al, 2007). Each Z-plane was 0.5 m. Background fluorescence was determined in three regions not containing cells throughout the entire z-stack.…”

Section: Methodsmentioning

confidence: 99%

“…Antibodies against caltubin were raised in rat against synthetic peptides corresponding to the N-terminal (MERAFEDVRSQHRD) and C-terminal (DG-KLSLDEFKTLYSP) regions of the protein. Cultured PeA neurons were fixed, permeabilized, and incubated with specific primary antibodies (NCS-1: 1:300, BIOMOL, catalog #BML-NL3750-0100; ␤-tubulin: 1:300, Sigma-Aldrich, catalog #T0198; ␣-tubulin: 1:300, Sigma-Aldrich, catalog #T6074; ␤-actin: 1:300, Sigma-Aldrich, catalog #A1978; caltubin: 1:200), as described previously (Gardzinski et al, 2007;Hui et al, 2007). Cells were then incubated with their respective secondary antibody (488 goat anti-rat, Millipore Bioscience Research Reagents; Alexa Fluor 568 goat anti-mouse, Invitrogen; Alexa Fluor 633 goat anti-rabbit, Invitrogen) for 2 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Caltubin, a Novel Molluscan Tubulin-Interacting Protein, Promotes Axonal Growth and Attenuates Axonal Degeneration of Rodent Neurons

Nejatbakhsh

Guo²,

Lu³

et al. 2011

J. Neurosci.

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Axotomized central neurons of most invertebrate species demonstrate a strong regenerative capacity, and as such may provide valuable molecular insights and new tools to promote axonal regeneration in injured mammalian neurons. In this study, we identified a novel molluscan protein, caltubin, ubiquitously expressed in central neurons of Lymnaea stagnalis and locally synthesized in regenerating neurites. Reduction of caltubin levels by gene silencing inhibits the outgrowth and regenerative ability of adult Lymnaea neurons and decreases local ␣-and ␤-tubulin levels in neurites. Caltubin binds to ␣-and/or ␤-tubulin in both Lymnaea and rodent neurons. Expression of caltubin in PC12 cells and mouse cortical neurons promotes NGF-induced axonal outgrowth and attenuates axonal retraction after injury. This is the first study illustrating that a xenoprotein can enhance outgrowth and prevent degeneration of injured mammalian neurons. These results may open up new avenues in molecular repair strategies through the insertion of molecular components of invertebrate regenerative pathways into mammalian neurons.

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“…As the two enhancers provided similar results, we present here data obtained by the Atoh1-enhancer. The brainstem and cerebellum were sectioned and stained with the synaptic markers synaptotagmin or SV2 (Gardzinski et al, 2007;Nowack et al, 2010), as well as with various neuronal markers, to allow the detection of dA1-synaptic target sites in correlation to other neurons along the embryonic brain.…”

Section: Synaptic Targets Of Da1 Interneuronsmentioning

confidence: 99%

Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain

Kohl

Hadas²,

Klar³

et al. 2012

J. Neurosci.

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Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is formed along the dorsal-most region of the caudal rhombic lip, gives rise to the cochlear and precerebellar nuclei. These centers project sensory inputs toward upper-brain regions. The fundamental role of dA1 interneurons in the assembly and function of these brainstem nuclei is well characterized. However, the precise en route axonal patterns and synaptic targets of dA1 interneurons are not clear as of yet. Novel genetic tools were used to label dA1 neurons and trace their axonal trajectories and synaptic connections at various stages of chick embryos. Using dA1-specific enhancers, two contralateral ascending axonal projection patterns were identified; one derived from rhombomeres 6 -7 that elongated in the dorsal funiculus, while the other originated from rhombomeres 2-5 and extended in the lateral funiculus. Targets of dA1 axons were followed at later stages using PiggyBac-mediated DNA transposition. dA1 axons were found to project and form synapses in the auditory nuclei and cerebellum. Investigation of mechanisms that regulate the patterns of dA1 axons revealed a fundamental role of Lim-homeodomain (HD) proteins. Switch in the expression of the specific dA1 Lim-HD proteins Lhx2/9 into Lhx1, which is typically expressed in dB1 interneurons, modified dA1 axonal patterns to project along the routes of dB1 subgroup. Together, the results of this research provided new tools and knowledge to the assembly of trajectories and connectivity of hindbrain dA1 interneurons and of molecular mechanisms that control these patterns.

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The role of synaptotagmin I C2A calcium‐binding domain in synaptic vesicle clustering during synapse formation

Cited by 23 publications

References 55 publications

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Caltubin, a Novel Molluscan Tubulin-Interacting Protein, Promotes Axonal Growth and Attenuates Axonal Degeneration of Rodent Neurons

Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain

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