(A and B) Anti-MBP immunostaining (green) of Schwann cell-DRG cocultures 6 days after addition of ascorbate in the presence of NGF (A) or the NGF scavenger TrkA-Fc (B). Scale bars equal 100 µm.
(C) Western blots of cocultures probed for myelin (P₀ and MAG) and Schwann cell (S100β) proteins. β-actin served as a loading control.
(D) Injection of TrkA-Fc (2.5 µg) along the mouse sciatic nerve on the day of birth and 2 days later reduced the expression of the myelin proteins P₀ and MAG at postnatal day 4 compared to the saline-injected contralateral nerve (protein levels assessed by Western blot, n = 16 per condition, % saline-injected control ± SEM, *p < 0.003, unpaired t test).

(A–C) p75^NTR is not required for the NGF-mediated enhancement of Schwann cell myelination. Dissociated DRG explants from p75^−/− mice fail to myelinate robustly without NGF (B), as determined by immunostaining for MBP (green) and Western blots (C) for myelin and Schwann cell proteins (P₀, MAG, and S100β). Scale bars equal 100 µm.
(D–F) Glial p75^NTR is not required for the NGF-mediated inhibition of oligodendrocyte myelination.
(D) No MBP-positive myelin (red) was observed in the presence of NGF in these xeno-cultures of rat DRGs with p75^NTR-null mouse OPCs (n = 6).
(E) Significant myelination was always seen in the presence of TrkA-Fc (n = 6).
(F) Higher-magnification view of p75^NTR-null myelinating oligodendrocytes.
(G and H) TrkA-activating antibodies (RTA, 50 µg/ml) are sufficient to mimic NGF in both types of myelinating cocultures. Anti-NGF neutralizing antibodies were added to block endogenous NGF, and mouse and rabbit IgG were used as controls for anti-NGF and RTA, respectively.
(G) Western blots of Schwann cell-DRG cocultures for Schwann cell and myelin proteins.
(H) Western blots of OPC-DRG cocultures for oligodendrocyte and myelin proteins.

(A and B) Anti-MBP immunostaining (red) of OPC-DRG cocultures 14 days after seeding OPCs in the presence of NGF (A) or the NGF scavenger TrkA-Fc (B). Scale bars equal 200 µm.
(C–E) A higher-magnification view of a myelinating oligodendrocyte in an OPC-DRG co-culture double-labeled with CC1 (C) and anti-MBP (D). Scale bars equal 100 µm.
(F) The presence of compact myelin in these cocultures was confirmed by electron microscopy.
(G) Western blots of OPC-DRG cocultures probed for myelin (MBP, MAG) and oligodendrocyte (CNPase) proteins indicate that NGF reduces oligodendrocyte maturation. β-actin served as a loading control.
(H–J) NGF also limits myelination by differentiated oligodendrocytes. Cocultures of DRGs seeded with acutely purified oligodendrocytes examined byanti-MBP immunostaining (red) after 3 days. Scale bars equal 200 µm.
(H) In the presence of NGF, many MBP-positive cells failed to extend processes, while others extended processes that failed to myelinate.
(I) In the presence of TrkA-Fc, many oligodendrocytes myelinated DRG axons only 3 days after seeding.
(J) Myelinating GalC⁺ A2B5⁻ oligodendrocytes were quantified by counting the proportion of MBP⁺ cells with at least two myelinating processes (16 fields/coverslip, 6 coverslips/condition, fold-change from NGF condition ± SD, *p < 0.001, unpaired t test).

(A–E) The effects of NGF on myelination require TrkA-positive DRGs.
(A and B) No effects of NGF or TrkA-Fc on the expression of myelin proteins in cocultures of Schwann cells (A) or cocultures of OPCs (B) with BDNF-dependent DRGs, as assessed by Western blot.
(C–E) No effects of NGF or TrkA-Fc on the numbers of oligodendrocytes or myelin internodes in cocultures of OPCs with BDNF-dependent DRGs, as assessed by immunostaining for MBP (red).
(F–H) Exposure of NGF to TrkA-expressing DRG soma or axons results in inhibition of oligodendrocyte myelination.
(F) TrkA-expressing DRGs in the somal compartment of Campenot chambers extend axons through a vacuum grease seal to the neighboring chamber. OPCs were seeded in the axonal chamber only.
(G) Extensive myelination (MBP; green) of TrkA (red)-expressing axons is only seen when TrkA-Fc is present in both the axonal and somal compartments.
(H) The number of nonmyelinating MBP-positive oligodendrocytes and myelin segments were quantified by counting 6 fields/chamber, 4 chambers/condition (cells or myelin segments/field ± SEM). *p < 0.01 versus NGF in both compartments and ^† p < 0.05 versus TrkA in both compartments (Student-Newman-Keuls post hoc comparison after one-way ANOVA).