Table 2.
Effects of Factors in Cell Culture on Neurite Outgrowth and Branching of Ciliary Ganglion Neurons at E8 Versus E13
| Factor | E8 Ciliary Ganglion
|
E13 Ciliary
|
E13 Choroid
|
|||
|---|---|---|---|---|---|---|
| #BP | NL (mm) | #BP | NL (mm) | #BP | NL (mm) | |
| Control | 7 ± 0.5 | 575 ± 20 | 2.3 ± 0.2 | 800 ± 35 | 2.9 ± 0.3 | 743 ± 82 |
| dTC | 9 ± 0.6 | 754 ± 33 | 1.6 ± 0.3 | 554 ± 91 | 2.4 ± 0.4 | 704 ± 66 |
| IGF-I | ND | ND | 1.8 ± 0.6 | 621 ± 89 | 2.0 ± 0.3 | 730 ± 90 |
| IGF-II | ND | ND | 1.7 ± 0.3 | 594 ± 60 | 2.5 ± 0.3 | 665 ± 50 |
| NT-3 | 9.4 ± 0.7 | 709 ± 35 | ND | ND | ND | ND |
| BDNF | 9.9 ± 0.6 | 736 ± 34 | 2.4 ± 0.3 | 694 ± 65 | 3.1 ± 0.4 | 832 ± 74 |
| TGFβ | 10 ± 0.7* | 846 ± 41* | 2.4 ± 0.5 | 800 ± 97 | 2.4 ± 0.4 | 673 ± 87 |
| NGF | 11.3 ± 0.7* | 882 ± 37* | 3.3 ± 0.4 | 926 ± 73 | 3.1 ± 0.5 | 598 ± 67 |
| GDNF | 11 ± 0.6* | 800 ± 35* | 3.8 ± 0.4+ | 795 ± 39 | 3.7 ± 0.5 | 734 ± 82 |
| CNTF | 11 ± 0.6* | 891 ± 61* | 5.7 ± 0.5+ | 1020 ± 76 | 4.9 ± 0.5⋄ | 805 ± 81 |
E8 or E13 neurons were isolated and grown on coverslips for 10 h (E8) or 21 h (E13) in the presence of the indicated growth factors. Neurites were labeled and the total length of all neurites (NL) per neuron as well as the number of branch points (BP) were quantified as described in methods. At E8, there are no markers for distinguishing ciliary from choroid neurons. At E13, choroid neurons were identified by somatostatin-like immunoreactivity (SOM-IR); ciliary neurons were negative for SOM-IR. Concentrations of growth factors were: 4.8 × 10−6M dTC, 10 ng/mL IGF-I, 10 ng/mL IGF-II, 10 ng/mL NT-3, 10 ng/mL BDNF, 10 ng/mL GDNF, 10 ng/mL TGF-β, 10 ng/mL CNTF, 1 ng/mL NGF (For E8), and 10 ng/mL NGF (for E13). Experiments were done in triplicate and repeated a minimum of three times. Statistical analysis was done by comparing BP and NO after addition of the individual neurotrophic factor to control with a one way ANOVA with Bonferroni post test. ND, not done. All values that were significantly different from untreated controls are bolded.
E8 neurons, n = 52–63 Branching: p < 0.01 for TGF-β, p < 0.001 for NGF, CNTF and GDNF; Outgrowth: p < 0.01 for GDNF, p < 0.001 for TGF-b, NGF, and CNTF.
E13 ciliary neurons, n = 23–143 #BP: p < 0.05 for GDNF, p < 0.01 for CNTF.
E13 choroid neurons, n = 33–144 #BP: p < 0.01 for CNTF.