Effects of moderate alcohol consumption on behavior and neural systems of Wistar rats.

Chronic alcohol consumption affects various neurotransmitters, especially those implicated in the transitioning to alcohol use disorders (particularly dopaminergic and CRFergic systems). Few studies have investigated moderate alcohol consumption and its harmful consequences. The objective of this work was to analyze behavioral and neurochemical (dopaminergic and CRFergic systems) alterations during chronic moderate alcohol consumption. Twelve male Wistar rats were submitted to an intermittent alcohol ingestion protocol (alcohol group) for four weeks. The control group consisted of six rats. Open Field and Elevated Plus Maze tests were used for analysis of motor and anxiety-like behaviors. Immunohistochemistry analysis was performed in dopaminergic and CRFergic systems. Animals exposed to alcohol consumed moderate doses, chronic and intermittently. Behavioral tests detected fewer fecal boli in the alcohol exposed group, and immunohistochemical analysis indicated fewer dopamine-immunoreactive cells in the ventral tegmental area, and more CRF-immunoreactive cells in the anterior cingulate cortex and dorsolateral septum in this group. Thus we concluded that Wistar rats that consumed moderate doses of alcohol voluntarily and chronically showed a discreet anxiolytic effect in behavior, and a hypodopaminergic and hyperCRFergic neurochemical condition, which together are strong inducers of alcohol consumption predisposing to the development of alcohol use disorder (AUD).

Estrogen therapy associated with mechanical vibration improves bone microarchitecture and density in osteopenic female mice.

We investigated the effects of estrogen therapy (ET) associated with low-intensity and high-frequency mechanical vibration (MV) on bone tissue in osteopenic female mice. Fifty 3-month-old female Swiss mice were ovariectomized (OVX) or sham-operated, and distributed after 4 months into the following groups, with 10 animals per group: Sham; Control, OVX + vehicle solution; MV, OVX + MV; ET, OVX + 17ß-estradiol; and MV + ET, OVX + MV and 17ß-estradiol. Both vehicle solution and 17ß-estradiol (10 µg kg-1  day-1 ) were injected subcutaneously 7 days per week, and vibration (0.6 g, 60 Hz) was delivered 30 min per day, 5 days per week. Bone mineral density (BMD) and body composition were evaluated by densitometry at baseline and after 60 days of treatment when the animals were euthanized, and their femurs underwent histomorphometric and histochemical analyses. The Control group showed increased weight and fat percentage, while the ET and MV + ET groups showed increased lean mass but decreased fat percentage. At the end of the treatment period, the BMD decreased in Control, remained constant in Sham and MV, and increased in ET and MV + ET. The MV + ET group showed the greatest bone volume compared with Sham (129%), Control (350%), MV (304%) and ET (14%). No differences occurred in cortical thickness. The Control group showed the highest content of mature collagen fibers, while the MV + ET group showed the highest content of immature collagen fibers. In conclusion, ET plus MV was effective in improving bone quality in osteopenic female mice, and this improvement is associated with specific changes in trabecular but not cortical bone.

Connexin37 deficiency alters organic bone matrix, cortical bone geometry, and increases Wnt/ß-catenin signaling.

Deletion of connexin (Cx) 37 in mice leads to increased cancellous bone mass due to defective osteoclast differentiation. Paradoxically; however, Cx37-deficient mice exhibit reduced cortical thickness accompanied by higher bone strength, suggesting a contribution of Cx37 to bone matrix composition. Thus, we investigated whether global deletion of Cx37 alters the composition of organic bone extracellular matrix. Five-month-old Cx37-/- mice exhibited increased marrow cavity area, and periosteal and endocortical bone surface resulting in higher total area in tibia compared to Cx37+/+ control mice. Deletion of Cx37 increased genes involved in collagen maturation (loxl3 and loxl4) and glycosaminoglycans- (chsy1, chpf and has3) proteoglycans- associated genes (biglycan and decorin). In addition, expression of type II collagen assessed by immunostaining was increased by 82% whereas collagen maturity by picrosirius-polarizarion tended to be reduced (p=0.071). Expression of glycosaminoglycans by histochemistry was decreased, whereas immunostaining revealed that biglycan was unchanged and decorin was slightly increased in Cx37-/- bone sections. Consistent with these in vivo findings, MLO-Y4 osteocytic cells silenced for Cx37 gene exhibited increased mRNA levels for collagen synthesis (col1a1 and col3a1) and collagen maturation (lox, loxl1 and loxl2 genes). Furthermore, mechanistic studies showed Wnt/ß-catenin activation in MLO-Y4 osteocytic cells, L5 vertebra, and authentic calvaria-derived osteocytes isolated by fluorescent-activated cell sorter. Our findings demonstrate that altered profile of the bone matrix components in Cx37-deficient mice acts in favor of higher resistance to fracture in long bones.

Defective cancellous bone structure and abnormal response to PTH in cortical bone of mice lacking Cx43 cytoplasmic C-terminus domain.

Connexin 43 (Cx43) forms gap junction channels and hemichannels that allow the communication among osteocytes, osteoblasts, and osteoclasts. Cx43 carboxy-terminal (CT) domain regulates channel opening and intracellular signaling by acting as a scaffold for structural and signaling proteins. To determine the role of Cx43 CT domain in bone, mice in which one allele of full length Cx43 was replaced by a mutant lacking the CT domain (Cx43(ΔCT/fl)) were studied. Cx43(ΔCT/fl) mice exhibit lower cancellous bone volume but higher cortical thickness than Cx43(fl/fl) controls, indicating that the CT domain is involved in normal cancellous bone gain but opposes cortical bone acquisition. Further, Cx43(ΔCT) is able to exert the functions of full length osteocytic Cx43 on cortical bone geometry and mechanical properties, demonstrating that domains other than the CT are responsible for Cx43 function in cortical bone. In addition, parathyroid hormone (PTH) failed to increase endocortical bone formation or energy to failure, a mechanical property that indicates resistance to fracture, in cortical bone in Cx43(ΔCT) mice with or without osteocytic full length Cx43. On the other hand, bone mass and bone formation markers were increased by the hormone in all mouse models, regardless of whether full length or Cx43(ΔCT) were or not expressed. We conclude that Cx43 CT domain is involved in proper bone acquisition; and that Cx43 expression in osteocytes is dispensable for some but not all PTH anabolic actions.

High bone mass in mice lacking Cx37 because of defective osteoclast differentiation.

Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37(-/-)) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37(-/-) mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37(-/-) mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37(+/+) littermates. sRANKL/M-CSF treatment of nonadherent Cx37(-/-) bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37(+/+) cell cultures. Further, Cx37(-/-) osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37(-/-) osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37(-/-) mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.