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Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A1R (CPA, CCPA) or A2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A1R (DPCPX) or A2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [3H]GUO binding became saturable at 100-300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [3H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [3H]GUO binding, but the sum of the two effects was able to displace [3H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A1R and A2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.

Previous study showed that low-intensity endurance exercise induced a significant recovery of damaged skeletal muscle in mdx mice, probably by reducing the degeneration of dystrophic muscle1. In order to explore the molecular basis of this observation, we perfomed a proteomic analysis to evaluate changes in proteins profiling of quadriceps dystrophic muscles of exercised versus sedentary mdx mice. Four protein spots were found significantly changed and were identified as three isoforms of Carbonic anhydrase 3 (CA3) and as superoxide dismutase [Cu-Zn] (SODC). Protein levels of CA3 isoforms were found significantly up-regulated in quadriceps of sedentary mdx mice (MDX-Sed) and were completely restored to wild type values in quadriceps of exercised mdx mice (MDX-Ex). Protein levels of SODC were found down-regulated in quadriceps of sedentary mdx mice and were significantly restored to wild type values in quadriceps of exercised mdx mice. These proteomic data, validated by Western blot analysis, indicate that low-intensity endurance exercise, by modulating some proteins involved in oxidative stress defense, may in part contribute to reduce the reported cell degeneration in mdx muscles1. Further investigations are needed to better define the extension of proteins change in MDX-Ex versus MDX-Sed mice and its correlation with the recovery of damaged fibers in MDX-Ex mice.

Mild endurance exercise training positively affects limb skeletal muscle in the mdx mice model of Duchenne Muscular Dystrophy (DMD). However, few and controversial data are available on the effects of mild exercise training on the diaphragm of mdx mice. The diaphragm was examined in mdx and wild type mice either under sedentary conditions (mdx-SD, WT-SD) or during mild exercise training (mdx-EX, WT-EX). At baseline and after 30 and 45 days of training (5 d/wk for 6 weeks), diaphragm muscle morphology and Cx39 protein were assessed. In addition, tissue levels of the chaperonin Hsp60 were measured at the same time points in gastrocnemius, quadriceps and diaphragm in each experimental group. Although morphological analysis showed unchanged total area of necrosis/regeneration in the diaphragm after training, there was a trend for regeneration areas to be larger than necrosis areas. However, the levels of Cx39 protein, a marker associated with active degeneration-regeneration process in damaged muscle were similar in the diaphragm of mdx-EX and mdx-SD mice. The diaphragm, but not limb muscles, of both trained and sedentary mdx mice showed decreased Hsp60 expression at 45 days, suggesting exhaustion of potentially protective mechanisms in the diaphragm similar to previous findings in lung epithelium. Compared to the positive effects of exercise training previously observed in limb skeletal muscles, the diaphragm showed little change after training.

The effects of mild endurance exercise training on the small airways in mdx mice are unknown. We compared epithelial thickness and turnover, apoptosis, and stress marker expression in small airways of mdx mice and wild-type (WT) controls, at rest and during exercise training. Mdx and WT mice were randomly assigned to sedentary (mdx-S, n=17; WT-S, n=19) or trained (mdx- EX, n=14; WT-EX, n=16) groups. Low-intensity endurance training (running on a wheel) was done 5 d/wk for 6 wk at progressively increasing speed (rpm from 16 to 24) and time (15 min to 1 h). Lungs were processed for light microscopy and periodic acid Schiff (PAS) staining. Hsp60 and PCNA were quantified by immunohistochemistry. Apoptosis was assessed by TUNEL. Bronchial epithelial thickness decreased over time in WT mice irrespective of training (linear regression for time trends: WT-S: R2=0.43, r= -0.65; WT-EX: R2=0.68, r= -0.82, p

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle weakness. We have previously shown that low-intensity endurance training prevented muscle damage (Frinchi et al, Int J Sports Med 2014). Since the effects of low-intensity endurance training on the the diaphragm in the mdx mouse model are unknown, in the same animals we investigated Cx39 protein levels (Western blotting) in homogenates of the diaphragm before and after training. Mdx and wild-type (WT) mice were randomly assigned to sedentary (mdx-S, n=17; WT-S, n=19) or trained (mdx-EX, n=14; WT-EX, n=16) groups. Low-intensity endurance training (running on a wheel) was done 5 days/week for 6 weeks at progressively increasing time (15 min to 1 h) and speed (rpm from 16 to 24, distance covered during training sessions from 48 to 288 m). Compared to our previous analysis of skeletal muscles changes in gastrocnemius and quadriceps, showing decreased muscle damage in trained vs sedentary mdx mice, analysis of protein level of Cx39 showed similar values in diaphragm homogenates from sedentary and trained mdx mice. These preliminary data suggest that prevention of muscle necrosis after mild training does not occur in the diaphragm. As a speculation, continuous work of diaphragm vs intermittent work of skeletal muscle might at least partly account for the different results obtained in respiratory and locomotor muscle.

The first aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) Cx37, Cx39, Cx40, Cx43, and Cx45 expression in developing skeletal muscle and in the adult regenerating skeletal muscle in response to crush injury. Among the Cxs examined, only the Cx39, Cx43 and Cx45 were found expressed during embryonic life and progressively reduced during early postnatal life to become dramatically expressed at very low levels like Cx43 and Cx45, or to be undetectable like Cx39 in the adult muscle. Cx37 and Cx40 were found expressed at low levels and were localized in the endothelial cells. In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers, or to repair the damaged one. Within 24h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesionforming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofibers. The second aim of this study was to examine how low-intensity endurance exercise affects the regeneration process in dystrophin-deficient skeletal muscle. The lack of dystrophin in mdx mice, an animal model for Duchenne muscular dystrophy, leads to cycles of muscle degeneration and regeneration processes. Male adult mdx and wild type mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 v Dott.ssa M. Frinchi days/week, for 4 weeks at progressively increasing loads. Exercised mdx mice showed a trend to lower body weight gain and positive effect on the degree of fatigue. Histomorphological analysis showed a significant reduction of both muscle necrosis foci and regeneration processes in the gastrocnemius and quadriceps muscles of exercised mdx mice. The reduction of regeneration process was also evaluated by examining the protein expression of Cx39, as a specific gene expressed during regeneration process of injured muscles. While Cx39 was not expressed both in wild type exercised nor in sedentary mice, it was markedly increased in sedentary mdx mice, because of active degeneration/regenerating process, and dropped to very low levels in exercised mdx mice, suggesting a reduction of muscle regeneration process. This study has shown that specific low intensity endurance exercise induces a strong beneficial effect on the regeneration of dystrophic muscle and may have therapeutic value at least to decrease progression of muscular dystrophy and in less extend for strengthening dystrophic muscle.

There are no clear data on the molecular mechanism by which the hippocampal 5-HT transmission contributes to the neuroprotective and antidepressant effects of 5-HT uptake blockers. Previously, we revealed that a 5-HT1A agonist may induce phosphorylation of FGFR1 and ERK1/2 in rat hippocampus independent of FGF-2, suggesting transactivation of FGFR1 tyrosine kinase in the absence of neurotrophic factor binding. As extension of previous work, using BRET analysis and coimmunoprecipitation in cellular models, FGFR1-5-HT1A heteroreceptor complexes have been demonstrated and agonist modulation characterized. In vitro assays on ERK1/2 phosphorylation in HEK cells and primary hippocampal cultures have shown synergistic increases in signaling upon coactivation with FGF-2 and 5-HT1A agonist. Coactivation of the FGFR1/5-HT1A heteroreceptor complex also resulted in synergistic increases in extensions of PC12 cells and neurite densities and protrusions in primary hippocampal cultures. The findings indicate that neurotrophic effects of 5-HT in brain may in part be mediated by activation of the 5-HT1A receptor in the hippocampal FGFR1/5-HT1A heteroreceptor complex enhancing the FGFR1 signaling and playing a significant role in hippocampal plasticity.

Cx36 and Cx45 are considered the main connexins expressed in neuronal cells of central nervous system (Belluardo N. et al. Brain Res. 2000)), but at present no complete data are available on their expression in the neurons of crest-derived neuroendocrine organ, such as the adrenal medulla, the carotid body and the enteric nervous system. Therefore the present study was undertaken to evaluate using immunohistochemistry and western blotting procedures, both in rat and mouse, whether Cx36 and Cx45 were expressed in neuronal cells of the carotid body, adrenal medulla and myenteric plexus at ileum and colon level. In the carotid body of both mouse and rat only Cx45 was found expressed as diffuse punctate immunostaining. High amount of Cx36 expression was detected in the medulla and zona glomerulosa of mouse adrenal gland, but not in the rat. The Cx45 was found only in the mouse zona reticularis of adrenal gland cortex. Very low amount of Cx36 punctate immunostaining was found in the myenteric plexus of mouse colon and ileum but not in the rat. No Cx45 immunostaining was evidenced in the myenteric plexus of both mouse and rat, although it was found along the circular muscle of colon and ileum. Using western blotting analysis we confirmed for both Cx36 and Cx45 the results obtained by immunohistochemistry. Taken together our data increase our insight into types of gap-junctions localized in neuronal of sympatho-adrenal system and their different expression between species.

Aim: The aim of present work was to test the hypothesis that Cx37, Cx39, Cx40, Cx43 and Cx45 expression could be regulated in adult regenerating skeletal muscle in response to injury promoting activation of satellite cells involved in myofibers repair and regeneration. Methods: Using in situ hybridization and immunohistochemistry procedures we examined the spatial and temporal expression pattern of above listed connexins in the regenerating gastrocnemious muscle following a mechanical injury. Results: Cx43 and Cx45 mRNA were up-regulated very early, by 3 hour following muscle injury, and were localised in satellite cells, M-cadherin positive cells, distributed around the area of lesion. Three days after lesion a large number of Cx43 and Cx45 mRNA labelled cells were found inside the area of lesion where they become myoblasts, myogenin positive, and participate to muscle regeneration. By contrast, Cx39, not expressed in control muscle, appears in myogenin positive cells 3 days following muscle lesion and its expression was restricted to the area of lesion. Cx39 mRNA was mainly localised in myoblast myogenin positive cells forming clusters or rows of closely apposed cell nuclei committed to form myotubes. Cx40 mRNA labelled cells were observed within 24 hours from injury in the endothelial cells of blood vessels around the area of lesion. After 3 days these cells were localised inside the area of lesion where they co-express myogenin and start to form myotubes. By contrast, Cx37 mRNA expression appeared by 24 hrs from lesion and never was colocalised in myogenin positive cells, but it was involved in new blood vessels formation. All transcripts examined reached a peak of expression between 7-9 days from injury and then progressively declined, being undetectable at 4-5 weeks from injury. Conclusion: Taken together these results support the suggestion that, in regenerating skeletal muscle several connexins may be differentially involved in communication of myogenic committed cells during the process of cell proliferation, aggregation and fusion to form new myotubes.