Y1 receptor (Y1R)-signalling pathway plays a pivotal role in the regulation of bone metabolism. tool (BonePit), showed that Y1R?/? resorption pits displayed a marked reduction in surface area, volume and depth. Together, these data demonstrates that the lack of Y1Rs stimulates the formation of larger multinucleated osteoclasts with reduced bone-resorbing activity, unveiling a novel therapeutic option for osteoclastic bone diseases based on Y1R-signalling ablation. Osteoclasts are highly specialized multinucleated cells involved in bone resorption that derive from precursor cells of the hematopoietic lineage1. Bone-resorbing osteoclasts act in a concerted manner with bone-forming osteoblasts to accomplish bone remodelling, where old and damaged bone is resorbed followed by the formation of new bone2. The exacerbation of osteoclast-resorbing activity observed in cases of metabolic bone diseases (e.g. osteoporosis, Pagets disease)3, metastatic bone disease4 and rheumatoid arthritis5, results in the formation of fragile bones, increased risk of fracture and disability, leading to serious pathological conditions and possibly to death. Hence, uncovering putative signalling pathways that control osteoclast activity is of vital importance and might unfold new promising treatment options. Traditionally, bone remodelling is viewed as a complex process regulated by hormonal, autocrine/paracrine and mechanical signals. However, Cyclosporin B growing evidence has shown that signalling molecules supplied by skeletal sympathetic and sensorial nerve fibbers might also be directly involved in the control of bone turnover through neurotransmitter receptors expressed by bone cells6. Among the neurotransmitters expressed in bone microenvironment, described to modulate bone mass, emerges the neuropeptide Y (NPY) system through Y1R Cyclosporin B signalling pathway7,8,9. Several findings supported a generalized and powerful peripheral action of Y1R signalling in the regulation of bone mass. Both germ line and osteoblast-specific Y1R deletion resulted in significantly greater cancellous and cortical bone volume in femoral, tibia, and vertebrae bones in mice7,8. Previous work from our group have also shown that the systemic blockade of Y1R signalling stimulates pronounced increments on bone mass10. These anabolic effects have been exclusively attributed to increased osteoblast activity, given that Y1Rs are expressed in differentiated osteoblasts11 and that the specific deletion of receptors from mature osteoblasts augmented osteoblast differentiation and activity12. Nonetheless, the involvement of Y1R signalling in bone turnover via osteoclast activity remains to be fully addressed. Y1R has been shown to be robustly expressed by immune cells (e.g. T-cells, monocytes and macrophages)13 and to play a pivotal role in macrophage function14. Since both osteoclasts and macrophages share a common progenitor lineage, these studies suggest a direct role for Y1Rs in osteoclast progenitors activity. Moreover, tartrate-resistant acid phosphatase Cyclosporin B (TRAP)-stained bone histological sections revealed an increase in osteoclast surface upon disruption of Y1R signalling in mice7,10. In the light of these indications, we hypothesize that Y1R signalling pathway might also be involved in the regulation of osteoclast activity and function, thus acting in both arms of bone remodelling. Hence, the aim of this study was to investigate the functional role of Y1R signalling in osteoclastogenesis and bone matrix resorption, using bone marrow-derived osteoclasts retrieved from wild-type (WT) and Y1R germ line knockout (Y1R?/?) mice. Moreover, we have also developed a novel computational tool C BonePit C that allowed to perform a detailed morphologic analysis of the resorption pits produced by osteoclasts in a 3D space. Results Y1R gene expression is upregulated during osteoclastogenesis Previous studies have demonstrated that Y1?/? mice retain a high-bone mass phenotype7. This feature has been associated with increased osteoblast activity and bone formation. Nevertheless little is known regarding the involvement of Y1R signalling in osteoclast function. Hence, we have first assessed the expression profile of Y1R Cyclosporin B during all stages of osteoclast differentiation15, using bone marrow retrieved from WT mice. The Y1R mRNA expression was demonstrated to be substantially upregulated throughout osteoclast differentiation (osteoclast fusion and maturation) by quantitative real-time PCR (qPCR) analysis (Fig. 1). As depicted in Fig. 1, Y1R gene expression increased considerably upon the differentiation of bone marrow-derived monocytes into small round mononucleated TRAP-positive cells (pre-osteoclasts) and the formation of multinucleated TRAP-positive cells (mature osteoclasts). These results provided the first indication that Y1Rs might play a regulatory function in osteoclast activity. Figure 1 Y1R gene expression profile during osteoclastogenesis. It is widely accepted that RANKL signalling pathway plays a role in osteoclast formation and differentiation16. Accordingly, the expression ACVR2 of RANKL and decoy receptor osteoprotegerin (OPG) was quantified by qRT-PCR analysis, using WT and Y1?/? osteoblast cultures (Supplementary Information Fig. S1). Quantitative analysis revealed that RANKL gene expression is elevated in Y1R?/? osteoblast cultures (p?