Promotion of dendritic growth by CPG15, an activity-induced signaling molecule

Science

Volumn 281, Issue 5384, 1998, Pages 1863-1866

  Nedivi, Elly ^a,b   Wu, Gang Yi ^a   Cline, Hollis T ^a  

^a Howard Hughes Medical Institute Cold Spring Harbor Laboratory Cold Spring Harbor   (United States)

^b MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BETA GALACTOSIDASE; CELL SURFACE PROTEIN; GLYCOSYLPHOSPHATIDYLINOSITOL; GROWTH PROMOTOR;

ANIMAL CELL; ARTICLE; CONTROLLED STUDY; DENDRITE; NERVE CELL GROWTH; NERVE PROJECTION; NONHUMAN; OPTIC TECTUM; PRIORITY JOURNAL; SIGNAL TRANSDUCTION; XENOPUS LAEVIS;

ANIMALS; DENDRITES; GENETIC VECTORS; GLYCOSYLPHOSPHATIDYLINOSITOLS; IMAGE PROCESSING, COMPUTER-ASSISTED; INTERNEURONS; LIGANDS; MEMBRANE PROTEINS; MICROSCOPY, CONFOCAL; NERVE TISSUE PROTEINS; NEURONAL PLASTICITY; NEURONS; RECOMBINANT PROTEINS; SIGNAL TRANSDUCTION; SUPERIOR COLLICULUS; VACCINIA VIRUS; XENOPUS LAEVIS;

ANIMALIA; XENOPUS LAEVIS;

EID: 0032544299     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5384.1863     Document Type: Article

References (39)

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4. Tissue samples from stage 46-48 tadpoles were homogenized as described [D.-J. Zou and H. T. Cline, Neuron 16, 529 (1996)] and size-separated on 15% SDS/tris-glycine gels before electroblotting onto nitrocellulose. Blots were incubated with a 1:100 dilution of unpurified anti-CPG15 or preimmune antisera and developed by ECL (Amersham). Polyclonal antiserum to CPG15 was generated in rabbits by Pocono Rabbit Farm and Laboratory against a FLAG fusion protein (Kodak) expressed in Escherichia coli BL21 and was purified by established methods [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)].
5. Tissue samples from stage 46-48 tadpoles were homogenized as described [D.-J. Zou and H. T. Cline, Neuron 16, 529 (1996)] and size-separated on 15% SDS/tris-glycine gels before electroblotting onto nitrocellulose. Blots were incubated with a 1:100 dilution of unpurified anti-CPG15 or preimmune antisera and developed by ECL (Amersham). Polyclonal antiserum to CPG15 was generated in rabbits by Pocono Rabbit Farm and Laboratory against a FLAG fusion protein (Kodak) expressed in Escherichia coli BL21 and was purified by established methods [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)].
6. KA was injected into the tadpole optic ventricle (50 μM). Intraperitoneal (ip) injection of KA into rats and subsequent removal of hippocampal dentate gyri were done as described (1).
7. E. Nedivi, A. Javaherian, H. T. Cline, in preparation.
8. E. Nedivi and H. T. Cline, unpublished data.
9. Stage 46-48 tadpoles were fixed in 4% paraformaldehyde, and their brains were dissected and cut into 30-μm horizontal cryostat sections. Sections were incubated with preimmune serum or antiserum to CPG15 at a 1:200 dilution and visualized with fluorescein isothiocyanate-tagged goat anti-rabbit (Sigma). Sections from virally infected animals were double-labeled with antiserum to CPG15 as above and antiserum to β-gal (Sigma) visualized with a Cy5-tagged secondary antibody.
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12. 8 plaque forming units), was mixed with 0.1% Fast Green, and 100 to 150 nl were injected into the tectal ventricle of anesthetized stage 46-48 [P. D. Nieuwkoop and J. Faber, Normal Table of Xenopus laevis (Daudin) (ELsevier-North Holland, Amsterdam, 1956)] albino Xenopus laevis tadpoles. After recovering from anesthesia, animals were kept at room temperature for 2 days, when tectal neurons were labeled and imaging began. β-gal immunohistochemistry showed that levels of infection ranged from 20 to 50% of tectal neurons and that both neurons and radial glia were infected.
13. 8 plaque forming units), was mixed with 0.1% Fast Green, and 100 to 150 nl were injected into the tectal ventricle of anesthetized stage 46-48 [P. D. Nieuwkoop and J. Faber, Normal Table of Xenopus laevis (Daudin) (ELsevier-North Holland, Amsterdam, 1956)] albino Xenopus laevis tadpoles. After recovering from anesthesia, animals were kept at room temperature for 2 days, when tectal neurons were labeled and imaging began. β-gal immunohistochemistry showed that levels of infection ranged from 20 to 50% of tectal neurons and that both neurons and radial glia were infected.
14. 8 plaque forming units), was mixed with 0.1% Fast Green, and 100 to 150 nl were injected into the tectal ventricle of anesthetized stage 46-48 [P. D. Nieuwkoop and J. Faber, Normal Table of Xenopus laevis (Daudin) (ELsevier-North Holland, Amsterdam, 1956)] albino Xenopus laevis tadpoles. After recovering from anesthesia, animals were kept at room temperature for 2 days, when tectal neurons were labeled and imaging began. β-gal immunohistochemistry showed that levels of infection ranged from 20 to 50% of tectal neurons and that both neurons and radial glia were infected.
15. 18(S) or 1,1′-dioctadecyl-3,3,3′3′-tert-methylindocarbocyanine perchlorate, Molecular Probes; 0.05% in absolute ethanol] using 1 to 10 nA positive current applied in three to five pulses of 1 to 10 ms duration. DiL was injected at different positions along the rostrocaudal axis of the tectum to label cells at a range of developmental stages. Mapping the positions of labeled cells within the tectum during the imaging sessions verified that injection sites for alt groups tested were within the same range along the rostrocaudal axis. Viral infections, dye labeling, screening, and imaging were done while animals were anesthetized with 0.02% 3-aminobenzoic acid ethyl ester (MS222, Sigma) in Steinberg's solution. Animals were screened for those with single or well-isolated brightly labeled tectal cells. The first image of each series was taken 1 to 2 hours after dye labeling.
16. Cells were imaged and analyzed as previously described (10). Cell drawings and measurements were done blind to the experimental treatment. Statistical significance was determined by two-tailed t-test.
17. Supporting documentation and figures are available via the Science Web site at www.sciencemag.org/ feature/data/982664.shl.
18. Cells with axons that were observed to exit the tectum were designated projection neurons. Interneurons were identified as neurons without an axonal projection exiting the tectum. Distinctions were possible because of the effort made during imaging to confirm the end point of any process extending beyond the range of the dendritic arbor (ambiguous cells were rejected from analysis). A second criterion to identify interneurons was their morphological similarity to previously defined interneurons of two types. One type has a pear-shaped cell body with a dendritic arbor similar to that of projection neurons and a short axon included within the dendritic field (Fig. 4). This type of cell is simiLar to interneurons described by M. Antal, N. Matsumoto, and G. Szekely [J. Comp. Neurol. 246, 238 (1986)]. The second type of interneuron is multipolar with a densely branched arbor (14). These cells resemble the T5(3) subclass of large asymmetric ganglionic neurons described as possible interneurons in a framework of lateral inhibition [N. Matsumoto, W. W. Schwippert, J.-P. Ewert, J. Comp. Physiol. 159, 721 (1986)].
19. Cells with axons that were observed to exit the tectum were designated projection neurons. Interneurons were identified as neurons without an axonal projection exiting the tectum. Distinctions were possible because of the effort made during imaging to confirm the end point of any process extending beyond the range of the dendritic arbor (ambiguous cells were rejected from analysis). A second criterion to identify interneurons was their morphological similarity to previously defined interneurons of two types. One type has a pear-shaped cell body with a dendritic arbor similar to that of projection neurons and a short axon included within the dendritic field (Fig. 4). This type of cell is simiLar to interneurons described by M. Antal, N. Matsumoto, and G. Szekely [J. Comp. Neurol. 246, 238 (1986)]. The second type of interneuron is multipolar with a densely branched arbor (14). These cells resemble the T5(3) subclass of large asymmetric ganglionic neurons described as possible interneurons in a framework of lateral inhibition [N. Matsumoto, W. W. Schwippert, J.-P. Ewert, J. Comp. Physiol. 159, 721 (1986)].
20. To identify neurons that were both labeled with Dil and infected with CPG15VV, we labeled cells with chloromethylated Dil (Molecular Probes) and imaged them in vivo, as described above. After two images were obtained (at 24-hour intervals), animals were fixed in 4% paraformaldehyde with 0.1% glutaraldehyde. Cell morphology was reconstructed from the images collected in vivo. TDBL and growth rate were determined as described (10). For those animals with neurons exhibiting the "outlier" phenotype, brains were dissected and sections were prepared as described (8). Sections were then incubated with monoclonal antibody to β-gal (Sigma) and subsequently with Cy5-tagged goat anti-mouse Fab fragment (Jackson). After immunostaining, the single Dil-labeled cell in each animal was identified, and images of the appropriate sections were collected at dual wavelengths on a Noran confocal microscope equipped with a krypton/argon laser to assess whether the Dil-labeled neurons (visualized at 488 nm) were immunoreactive for β-gal (visualized at 647 nm).
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39. We thank B. Burbach, K. Bronson, I. Miloslavskaya, and N. Dawkins for excellent technical assistance, Z. Li for making the pSC65-CPG15t3 construct, and R. Malinow, K. Svoboda, and J. Yin for critical reading of the manuscript. Supported by NIH (H.T.C. and E.N.), the National Down Syndrome Society (H.T.C.), and the Marie Robertson Fund (E.N.).