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ProSAAS is one of the most abundant proteins in the brain and is processed into several smaller peptides. One of which, BigLEN, is an endogenous ligand for the G protein-coupled receptor, GPR171. Recent work in rodent models has shown that a small-molecule ligand for GPR171, MS15203, increases morphine antinociception and is effective in lessening chronic pain. While these studies provide evidence for GPR171 as a possible pain target, its abuse liability has not yet been assessed which we evaluate in the current study. We first mapped the distribution of GPR171 and ProSAAS throughout the reward circuit of the brain using immunohistochemistry and showed that GPR171 and ProSAAS are localized in the hippocampus, basolateral amygdala, nucleus accumbens, prefrontal cortex. In the major dopaminergic structure, the ventral tegmental area (VTA), GPR171 appeared to be primarily localized in dopamine neurons while ProSAAS is outside of dopamine neurons. Next, MS15203 was administered to mice with or without morphine, and VTA slices were stained for the immediate early gene c-Fos as a marker of neuronal activation. Quantification of c-Fos-positive cells revealed no statistical difference between MS15203 and saline, suggesting that MS15203 does not increase VTA activation and dopamine release. Similarly, the results of a conditioned place preference experiment showed that treatment with MS15203 or MS15203 + Morphine produced no place preference compared to Saline indicating a lack of reward-related behavior. Taken together this data provides evidence that the novel pain therapeutic, MS15203, has minimal reward liability. Therefore, GPR171 deserves further exploration as a pain target.Significance StatementMS15203, a drug that activates the receptor GPR171, was previously shown to increase morphine analgesia. The authors use in vivo and histological techniques to show that it fails to activate the rodent reward circuitry, providing support for the continued exploration of MS15203 as a novel pain drug, and GPR171 a novel pain target.

Intervertebral disc degeneration (IVDD) is linked to low back pain. Microstructural changes during degeneration have previously been imaged using 2D sectioning techniques and 3D methods which are limited to small specimens and prone to inducing artefacts from sample preparation. This study explores micro computed X-ray tomography (microCT) methods with the aim of resolving IVD 3D microstructure whilst minimising sample preparation artefacts. Low X-ray absorption contrast in non-mineralised tissue can be enhanced using staining and phase contrast techniques. A step-wise approach, including comparing three stains, was used to develop microCT for bovine tail IVD using microfocus tube (‘laboratory’) and synchrotron sources. Staining successfully contrasted collagenous structures; however not all regions were stained and the procedure induced macroscopic structural changes. Phase contrast microCT of chemically fixed yet unstained samples resolved the nucleus pulposus, annulus fibrosus and constituent lamellae, and finer structures including collagen bundles and cross-bridges. Using the same imaging methods native tissue scans were of slightly lower contrast but free from sample processing artefacts. In the future these methods may be used to characterise structural remodelling in soft (non-calcified) tissues and to conduct in situ studies of native loaded tissues and constructs to characterise their 3D mechanical properties.

Objective To explore whether aberrant transient chondrocyte behaviors occur in the joints of STR/Ort mice (which spontaneously develop osteoarthritis [OA]) and whether they are attributable to an endochondral growth defect. Methods Knee joints from STR/Ort mice with advanced OA and age-matched CBA (control) mice were examined by Affymetrix microarray profiling, multiplex polymerase chain reaction (PCR) analysis, and immunohistochemical labeling of endochondral markers, including sclerostin and MEPE. The endochondral phenotype of STR/Ort mice was analyzed by histologic examination, micro-computed tomography, and ex vivo organ culture. A novel protocol for quantifying bony bridges across the murine epiphysis (growth plate fusion) using synchrotron x-ray computed microtomography was developed and applied. Results Meta-analysis of transcription profiles showed significant elevation in functions linked with endochondral ossification in STR/Ort mice (compared to CBA mice; P < 0.05). Consistent with this, immunolabeling revealed increased matrix metalloproteinase 13 (MMP-13) and type X collagen expression in STR/Ort mouse joints, and multiplex quantitative reverse transcriptase-PCR showed differential expression of known mineralization regulators, suggesting an inherent chondrocyte defect. Support for the notion of an endochondral defect included accelerated growth, increased zone of growth plate proliferative chondrocytes (P < 0.05), and widespread type X collagen/MMP-13 labeling beyond the expected hypertrophic zone distribution. OA development involved concomitant focal suppression of sclerostin/MEPE in STR/Ort mice. Our novel synchrotron radiation microtomography method showed increased numbers (P < 0.001) and mean areal growth plate bridge densities (P < 0.01) in young and aged STR/Ort mice compared to age-matched CBA mice. Conclusion Taken together, our data support the notion of an inherent endochondral defect that is linked to growth dynamics and subject to regulation by the MEPE/sclerostin axis and may represent an underlying mechanism of pathologic ossification in OA.