Fig 2: Co-localization of CD40 and CD40L in the cultured hippocampal pyramidal neurons and striatal MSNs.(A) Representative images of dissociated E18 hippocampal cultures after 9 days in vitro triple labelling with anti-CD40, anti-CD40L and DAPI. The upper panels show images of neuronal cell bodies and proximal neural processes. The lower panels show distal neural processes. (B) Representative images of dissociated E14 striatal cultures after 10 days in vitro triple labelling with anti-CD40, anti-CD40L and βIII tubulin. Scale bar, 20 μm.

Fig 3: CD40-activated CD40L reverse signalling reduces neurite growth from cultured MSNs.(A) Representative photomicrographs of MSNs of Cd40+/+ and Cd40-/- E14 embryos cultured for 10 days and treated with either soluble CD40L (sCD40L) at 0.5 μg/ml, CD40-Fc or Fc protein (each at 1 μg/ml) or untreated (control). Neurons were double labelled for βIII tubulin (red) and DARPP-32 (green) to identify MSNs. Scale bar, 50 μm. (B) Quantification of total neurite lengths of MSNs after 10 days in vitro. Mean ± s.e.m of data collected from three independent experiments from Cd40+/+ (control, n = 69; sCD40L, n = 65; Fc, n = 63; CD40-Fc, n = 62) and Cd40-/- (control, n = 60; sCD40L, n = 62; Fc, n = 52; CD40-Fc, n = 73). One-way ANOVA with multiple Newman-Keuls statistical comparisons with control. ***p<0.0001 (actual p values provided in the text).10.7554/eLife.30442.014Figure 4—source data 1.Means, s.e.m. and n numbers for bar charts.

Fig 4: Localization of CD40 and CD40L in the developing hippocampus.Representative images of sections of the CA1 region of P9 hippocampi labelled with either anti-CD40 (A) or anti-CD40L (B) together with DAPI and either anti-MAP-2 or anti-GFAP (pl, pyramidal layer; sr, stratum radiatum; slm, stratum lacunosum moleculare). (C) Antibody specificity controls: sections of the hippocampus of P9 Cd40-/- mice incubated with the anti-CD40 antibody and sections of the hippocampus of P9 Cd40+/+ mice that received no primary antibody. Scales bar, 50 μm.

Fig 5: CD40-activated CD40L reverse signalling enhances dendrite growth from cultured hippocampal pyramidal neurons.(A) Representative photomicrographs of hippocampal neurons of Cd40+/+ and Cd40-/- E18 embryos cultured for 9 days and treated 24 hr after plating with either soluble CD40L (sCD40L), CD40-Fc or Fc protein (each at 1 μg/ml) or untreated (control). Scale bar, 100 μm. (B) Quantification of total dendrite lengths of neurons in these cultures after 9 days in vitro. Mean ± s.e.m of data collected from three independent experiments from Cd40+/+ (control, n = 70; sCD40L, n = 51; Fc, n = 55; CD40-Fc, n = 61) and Cd40-/- (control, n = 60; sCD40L, n = 57; Fc, n = 61; CD40-Fc, n = 59). One-way ANOVA with multiple Newman-Keuls statistical comparison with control, *p<0.01, and ***p<0.0001 (actual p values provided in the text).10.7554/eLife.30442.010Figure 2—source data 1.Means, s.e.m. and n numbers for bar charts.