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doi: 10.1242/10.1242/dev.00565

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Crucial role of TrkB ligands in the survival and phenotypic differentiation of developing locus coeruleus noradrenergic neurons

Pontus C. Holm1, Francisco J. Rodríguez1, Adelheid Kresse1,*, Josep M. Canals1,{dagger}, Inmaculada Silos-Santiago2 and Ernest Arenas1,{ddagger}

1 Department of Medical Biochemistry and Biophysics, Laboratory of Molecular Neurobiology, Karolinska Institutet, Stockholm S-171 77, Sweden
2 Millennium Pharmaceuticals Incorporated, 75 Sidney Street, Cambridge, MA 02139, USA



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  		Fig. 1. Developmental regulation of TrkB (A-A'), TrkC (A), Ret (B-B'), GFR{alpha}1 (B) and GFR{alpha}2 (B) mRNA expression in the mouse LC, as assessed by in situ hybridization with 35S-labeled riboprobes from embryonic day (E) 13 to postnatal day (P) 0. Dark-field pictures in A' and B' show TrkB and Ret hybridization, respectively. Note the intense Ret signal in the mesencephalic nucleus of the trigeminus, in the lower-left corner of B'. High magnification bright field pictures of representative cells of the main dark field pictures are presented in the upper-left corner of A' and B'. Adjacent sections hybridized with a DIG-labeled TH riboprobe were used to identify the position of the LC (bright field pictures in A'' and B''). The hybridization signal for each of the 35S-labeled probes was measured using NIH Image in: (1) the area occupied by the LC; (2) in the vicinity of the LC to control for background signal in the tissue; (3) in the tissue-free area of the fourth ventricle. The background value of the tissue-free area was subtracted from the labeling values in the LC area and the non-labeled control tissue, before statistical comparison and graph plotting. Values represent the mean±s.e.m. of 4 measures per section in 3 sections per animal in 3 animals per condition. Statistical analysis of the in situ signal showed that all time points in A and B, except for the GFR{alpha}1 signal at E15 and the TrkC signal at P0, signals were significantly higher than the tissue background signal (P<0.05, paired t-test). Scale bar: 100 µm.

 


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  		Fig. 2. Gdnf/Nt3 double null mutant mice do not differ from wild-type mice in LC cell numbers, expression of phenotypic markers or target innervation. The LC of wild-type (A,E-L), Gdnf null mutant (B), Nt3 null mutant (C) and Gdnf/Nt3 double null mutant (D,E'-L') were analyzed at P0. The number of LC NA neurons, assessed by in situ hybridization with riboprobes against DBH mRNA in wild-type, Gdnf

-/-, Nt3-/- and Gdnf/Nt3 double null mutants, did not differ (A-D). Moreover, immunohistochemistry for TH (E,E') and NOS (F,F') and in situ hybridization mRNA detection of BDNF (G,G'), NPY (H,H'), Galanin (I,I') and CGRP (J,J') did not differ in wild-type and Gdnf/Nt3 double null mutant mice. Finally, retrograde DiI tracing from the spinal cord (K,K') and nucleus accumbens (L,L') showed that the innervation of target structures in double null mutant mice does not differ from that in the wild-type mice.

 


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  		Fig. 3. Neither GDNF nor NT3 increases the number of TH-immunoreactive noradrenergic neurons in E13.5 rat LCr primary cultures. Cells were treated with 30 ng/ml GDNF and/or 30 ng/ml NT3 and were analyzed after 6 days in vitro. Cell counts revealed no significant differences in the number of TH-positive cells between control wells and wells treated with GDNF, NT3 or GDNF+NT3.

 


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  		Fig. 4. Loss of TH-positive and CV-positive LC NA neurons in the TrkB null mutant mice. A-D show representative sections through the LC of wild-type (A), TrkB-/- (B), TrkC-/- (C) and TrkB-/- TrkC+/- (D) at P0 after TH immunohistochemistry. The graph in E describes the average number of TH-positive cells (±s.d.) in the LC of wild-type, TrkB+/-, TrkB-/-, TrkC-/- and TrkB-/- TrkC+/- mice. TrkB-/- was significantly different from wild-type and TrkB+/- (*P<0.05), and TrkB-/- TrkC+/- was different from TrkB+/- (#P<0.05) as assessed by unpaired t-test (n=4-10). The number of TH-positive and CV-positive neurons in the LC was examined in adjacent sections of wild-type and TrkB null mutant mice at P0 (F). A decrease of 28.3% and 25.7% in the number of TH-positive and CV-positive neurons, respectively, was detected in the TrkB-/- mice. (*P<0.05, paired t-test). Scale bar: 100 µm.

 


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  		Fig. 5. BDNF or NT4 increases the number of LCr NA neurons after 6 days in vitro. Dissociated rat E13.5 LCr primary cultures were treated as indicated on the x-axis. (A) Cell counts showed a tenfold increase in number of TH-positive cells for all conditions in which a TrkB ligand is present (*P<0.001, unpaired t-test). B-D show representative fields of LCr cultures with TH-positive cells after treatment with control media (B, N2) or N2 with BDNF (C) or NT4 (D). Scale bar: 100 µm.

 


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  		Fig. 6. The increase in the number of TH-positive cells in LC cultures does not involve proliferation but rather survival and a second mechanism. In A, a representative TH-positive cell (red cytoplasm) and a BrdU-positive cell (dark nucleus) from an E13.5 LCr culture treated with BrdU and BDNF for 24 hours is shown. TH-positive cells in the culture were negative for BrdU and the number of BrdU-positive cells did not increase by BDNF or NT4 treatment. The graph in B displays the number of TH-positive cells after treatment with BDNF, NT4, NT3, bFGF or control media for 2, 24 or 48 hours. A steep increase in the number of TH-positive cells was detected between 2 and 24 hours in BDNF and NT4-treated cultures, whereas bFGF only induced a significant increase in cell number after 48 hours (*P<0.01, #P<0.05, unpaired t-test). (C) When NT4 was added to E13.5 LC cells before tissue dissociation (NT4 early), a constant higher number of TH-positive cells were detected in the wells, at all stages analyzed, as compared to wells in which NT4 was added at the time of plating (NT4 late), suggesting that early administration of NT4 prevents the loss of a population of TH-positive cells that would otherwise die shortly after dissociation. Between 2 and 12 hours a parallel increase in the number of noradrenergic neurons was detected in both early (at the time of dissociation) and late (at the time of plating) NT4 treatment, indicating that an additional mechanism is involved. *P<0.01 with respect to control and P<0.05 with respect to late NT4 treatment, #P<0.01 with respect to control, by unpaired t-test. Scale bar: 50 µm.

 


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  		Fig. 7. Delayed administration of NT4 for 24 hours but not 48 hours increases the number of TH-positive cells in LCr cultures. NT4 was added to E13.5 LCr cultures at the time of plating or after 24 or 48 hours and then the cultures were fixed after 24, 48 or 72 hours. Counts of TH immunoreactive cells revealed that addition of NT4 after 24 hours gave the same number of TH-positive cells than at the time of plating, suggesting a second effect of NT4 distinct from the early survival-promoting effect. *P<0.01, unpaired t-test.

 


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  		Fig. 8. BDNF or NT4 selectively increased the number of Phox2a-positive cells that co-express TH. (A) BDNF increased the number of TH and Phox2a double positive cells between 3 and 6 hours in culture. This effect was maintained for up to 6 days in vitro (B). (B) Administration of either BDNF or NT4 selectively increased the pool of Phox2a-positive/TH-positive cells after 6 days in vitro, leaving the pool of Phox2a-positive/TH-negative cells unaffected. *P<0.05, unpaired t-test.

 

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