a, Bone mineral density at the spine and femur assessed by DXA in CB₁ KO mice and wild type littermates; b trabecular bone mineral density at the tibial metaphysis assessed by pQCT in CB₁ KO mice and wild type littermates; c, representative photomicrograph of the proximal tibia from and CB₁ KO mice (left panel) and wild type mice (right panel) Values in the bar charts are means and standard errors. Significant differences between CB₁ KO and wild type mice are indicated by: ***p<0.001; *p<0.02.

a, Effects of the CB ligands on osteoclast formation in C57BL/6 bone marrow cultures stimulated with RANKL and M–CSF. The data shown are from three independent experiments and are expressed as a percent of values in control cultures.. Significant inhibition or stimulation of osteoclast formation when compared with control is indicated by * p<0.05 or below. b, representative photomicrographs of vehicle treated culture and cultures treated with anandamide (AEA), SR144528 and AM251 stained for TRAcP. Osteoclasts (arrows) were larger and more numerous in the AEA treated cultures when compared with vehicle and were virtually absent from the SR144528 and AM251 treated cultures. c, effect of AM251 alone (1μM) and AM251 in combination with AEA (5μM) on osteoclast formation. Values in the bar charts and line graphs are means and standard deviations. Significant differences are indicated by: *** p<0.001 (vehicle vs. AM251); # p<0.02 (AM251 vs. AM251 + AEA).

a. Effects of AM251 on trabecular bone density in sham operated (sham) and ovariectomized (ovx) C57BL/6 mice; b, Effects of AM251 on trabecular number in sham operated (sham) and ovariectomized (ovx) C57BL/6 mice. Values are expressed as percent change relative to sham operated vehicle treated control mice. c, representative photomicrograph of the tibial metaphysis 21 days after ovariectomy in a vehicle treated mouse (top panel) and an AM251 treated mouse (bottom panel). Values in the bar charts are means and standard errors. Significant differences between sham Ovx and sham Ovx + AM251 and between sham Ovx and Ovx are indicated by ** p<0.001; significant differences between Ovx and Ovx + AM251 are indicated by ## p<0.002.

a, Total BMD at the tibial metaphysis in CB₁ KO mice and wild type littermates before and after sham operation or ovariectomy (ovx); b, Bone volume / total volume (BV/TV) assessed at the same site by μCT; c, Trabecular thickness (Tb.Th) assessed by μCT; d trabecular number (Tb.N) assessed by μCT. Values in the bar charts are expressed as the percent change relative to the value in sham operated wild type animals and are means and standard errors. Significant differences between CB₁ KO and wild type mice are indicated by: *** p<0.001; ** p<0.01.

a, effect of AM251 on osteoclast formation in RANKL / M–CSF stimulated bone marrow cultures prepared from CB₁ KO mice and wild type littermates. b, effect of AM630 on osteoclast formation in bone marrow mononuclear cells cultured from wild type and CB₁ KO mice. Values are means and standard deviations. Significant differences from control cultures are indicated by * p<0.05. Significant differences between CB₁ KO and wild type cultures are indicated by # p<0.05.

a, Effects of the CB antagonist AM251 (5μM) on ERK phosphorylation as detected by western blot in vehicle (C) and RANKL (100ng/ml) treated mouse osteoclasts. The numbers refer to the time (min) after RANKL stimulation. MW is molecular weight marker and the size of the marker bands is indicated in kDa. b, Effects of AM251 on nuclear translocation and DNA binding of phospho NFATc1 in mouse osteoclasts treated with RANKL (100ng/ml) in the presence of vehicle or AM251 (2μM). c, Effects of AM251 on nuclear translocation and DNA binding of c–jun in mouse osteoclasts treated with RANKL (100ng/ml) in the presence of vehicle or AM251 (2μM). d, Effects of AM251 on DNA binding of c–fos in mouse osteoclasts treated with RANKL (100ng/ml) in the presence of vehicle or AM251 (2μM). Values are expressed as the change relative to vehicle treated cultures. Values in the bar charts are means and standard deviations. Significant differences between vehicle and RANKL treated cultures are indicated by: *** p<0.001; and differences between AM251 and vehicle treated cultures are indicated by: ## p<0.001. The results shown are representative of three independent experiments.