Fig 2: CD40–CD40L signaling modulates NGF‐promoted axon growth over the same developmental window in PVG and SCG neurons. Sholl plots PVG (a–d) and SCG (e–h) neurons at E18 (a,e), P0 (b,f), P3 (c,g) and P5 (d,h) Cd40 +/+ and Cd40 –/– littermates grown for 24 hr with 1 ng/ml of NGF. The insets show bar charts of axon length for each age. Mean ± SEM data of >150 neurons per condition combined from three experiments of each type. ***p < .001, *p < .05 (the differences between both length and branch points of Cd40+/+ and Cd40 –/– PVG and SCG neurons were analyzed by Mann–Whitney test)

Fig 3: CD40–CD40L signaling has opposing effects on NGF‐promoted axon growth from PVG and SCG neurons. Total axon length (a,e), branch point number (b,f), Sholl plots (c,g) and representative images (d,h) of the neurite arbors of PVG (a–d), and SCG (e–h) neurons of P0 Cd40 +/+ and Cd40 –/– littermates grown for 24 hr with 1 ng/ml of NGF. Mean ± SEM data of >150 neurons per condition combined from three experiments of each type. Scale bar, 50 μm. ***p < .001, *p < .05 (the differences between both length and branch points of Cd40+/+ and Cd40 –/– PVG and SCG neurons were analyzed by Mann–Whitney test) [Color figure can be viewed at https://www.wileyonlinelibrary.com]

Fig 4: PVG and SCG neurons co‐express CD40 and CD40L. Representative images of P0 PVG neurons (a,b) and SCG neurons (c,d) cultured for 24 hr in medium containing 1 ng/ml of NGF and double labeled for either anti‐CD40 and anti‐βIII tubulin (a,c) or anti‐CD40L and anti‐βIII tubulin (b,d). The preparations were also counterstained with the nuclear marker DAPI. Scale bar, 10 μm [Color figure can be viewed at https://www.wileyonlinelibrary.com]

Fig 5: Regulation of Cd40 mRNA expression in PVG and SCG neurons by NGF. Bar chart of the levels of Cd40 mRNA relative to reference mRNAs in wild‐type cultures of P0 PVG and SCG neurons cultured for 24 hr with and without 10 ng/ml of NGF. The data are normalized to a value of 1.0 for PVG neurons and for SCG neurons cultured without NGF. All cultures received 25 mM Boc‐D‐FMK. Mean ± SEM of data from three separate experiments