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BDNF signaling in hypothalamic circuitries regulates mammalian food intake. However, whether BDNF exerts metabolic effects on peripheral organs is currently unknown. Here, we show that the BDNF receptor TrkB.T1 is expressed by pancreatic β-cells where it regulates insulin release. Mice lacking TrkB.T1 show impaired glucose tolerance and insulin secretion. β-cell BDNF-TrkB.T1 signaling triggers calcium release from intracellular stores, increasing glucose-induced insulin secretion. Additionally, BDNF is secreted by skeletal muscle and muscle-specific BDNF knockout phenocopies the β-cell TrkB.T1 deletion metabolic impairments. The finding that BDNF is also secreted by differentiated human muscle cells and induces insulin secretion in human islets via TrkB.T1 identifies a new regulatory function of BDNF on metabolism that is independent of CNS activity. Our data suggest that muscle-derived BDNF may be a key factor mediating increased glucose metabolism in response to exercise, with implications for the treatment of diabetes and related metabolic diseases., Glucose metabolism is regulated by hypothalamic brain functions and factors produced by peripheral tissues. Here, the authors show that the regulator of food intake Brain-derived neurotrophic factor is also produced and secreted by muscle and stimulates pancreas insulin release.

The central nervous system has long been thought to regulate insulin secretion, an essential process in the maintenance of blood glucose levels. However, the anatomical and functional connections between the brain and insulin-producing pancreatic β-cells remain undefined. Here, we describe a functional transneuronal circuit connecting the hypothalamus to β-cells in mice. This circuit originates from a subpopulation of oxytocin neurons in the paraventricular hypothalamic nucleus (PVN(OXT)) and via the sympathetic autonomic branch it reaches the islets of the endocrine pancreas to innervate β-cells. Stimulation of PVN(OXT) neurons rapidly suppresses insulin secretion and causes hyperglycemia. Conversely, silencing of these neurons elevates insulin levels by dysregulating neuronal signaling and secretory pathways in β-cells and induces hypoglycemia. PVN(OXT) neuronal activity is triggered by glucoprivation. Our findings thus reveal that a subset of PVN(OXT) neurons form functional multisynaptic circuits with β-cells in mice to regulate insulin secretion and their function is necessary for the β-cell response to hypoglycemia.

Recent studies have suggested that the use of cognitive enhancers as adjuncts to exposure-based therapy in individuals suffering from post-traumatic stress disorder (PTSD) may be beneficial. Brain cholinergic signaling through basal forebrain projections to the hippocampus is an established pathway mediating fear response and cognitive flexibility. Here we employed a genetic strategy to enhance cholinergic activity through increased signaling of the NGF receptor TrkA. This strategy leads to increased levels of the marker of cholinergic activation, acetylcholine synthesizing enzyme choline acetyltransferase, in forebrain cholinergic regions and their projection areas such as the hippocampus. Mice with increased cholinergic activity do not display any neurobehavioral abnormalities except a selective attenuation of fear response and lower fear expression in extinction trials. Reduction in fear response is rescued by the GABA antagonist picrotoxin in mutant mice, and, in wild-type mice, is mimicked by the GABA agonist midazolam suggesting that GABA can modulate cholinergic functions on fear circuitries. Importantly, mutant mice also show a reduction in fear processing under stress conditions in a single prolonged stress (SPS) model of PTSD-like behavior, and augmentation of cholinergic signaling by the drug donepezil in wild-type mice promotes extinction learning in a similar SPS model of PTSD-like behavior. Donepezil is already in clinical use for the treatment of dementia suggesting a new translational application of this drug for improving exposure-based psychotherapy in PTSD patients.

Obesity is the key driver of peripheral insulin resistance, one of the key features of type 2 diabetes (T2D). In insulin-resistant individuals, the expansion of beta-cell mass is able to delay or even prevent the onset of overt T2D. Here, we report that beta-arrestin-1 (barr1), an intracellular protein known to regulate signaling through G protein-coupled receptors, is essential for beta-cell replication and function in insulin-resistant mice maintained on an obesogenic diet. Specifically, insulin-resistant beta-cell-specific barr1 knockout mice display marked reductions in beta-cell mass and the rate of beta-cell proliferation, associated with pronounced impairments in glucose homeostasis. Mechanistic studies suggest that the observed metabolic deficits are due to reduced Pdx1 expression levels caused by beta-cell barr1 deficiency. These findings indicate that strategies aimed at enhancing barr1 activity and/or expression in beta-cells may prove useful to restore proper glucose homeostasis in T2D., During insulin-resistance, the compensatory expansion of beta-cell mass is able to delay or the onset of overt type 2 diabetes. Here, the authors report that beta-arrestin-1, an intracellular protein known to regulate signalling through G protein-coupled receptors, is essential for beta-cell replication and function in insulin-resistant mice.

The pathophysiology of Amyotrophic Lateral Sclerosis (ALS), a disease caused by the gradual degeneration of motoneurons, is still largely unknown. Insufficient neurotrophic support has been cited as one of the causes of motoneuron cell death. Neurotrophic factors such as BDNF have been evaluated in ALS human clinical trials, but yielded disappointing results attributed to the poor pharmacokinetics and pharmacodynamics of BDNF. In the inherited ALS G93A SOD1 animal model, deletion of the BDNF receptor TrkB.T1 delays spinal cord motoneuron cell death and muscle weakness through an unknown cellular mechanism. Here we show that TrkB.T1 is expressed ubiquitously in the spinal cord and its deletion does not change the SOD1 mutant spinal cord inflammatory state suggesting that TrkB.T1 does not influence microglia or astrocyte activation. Although TrkB.T1 knockout in astrocytes preserves muscle strength and co-ordination at early stages of disease, its specific conditional deletion in motoneurons or astrocytes does not delay motoneuron cell death during the early stage of the disease. These data suggest that TrkB.T1 may limit the neuroprotective BDNF signaling to motoneurons via a non-cell autonomous mechanism providing new understanding into the reasons for past clinical failures and insights into the design of future clinical trials employing TrkB agonists in ALS.

The slow afterhyperpolarising current, sIAHP, is a Ca2+-dependent current that plays an important role in the late phase of spike frequency adaptation. sIAHPis activated by voltage-gated Ca2+channels, while the contribution of calcium from ryanodine-sensitive intracellular stores, released by calcium-induced calcium release (CICR), is controversial in hippocampal pyramidal neurons. Three types of ryanodine receptors (RyR1-3) are expressed in the hippocampus, with RyR3 showing a predominant expression in CA1 neurons. We investigated the specific role of CICR, and particularly of its RyR3-mediated component, in the regulation of the sIAHPamplitude and time course, and the activity-dependent potentiation of the sIAHPin rat and mouse CA1 pyramidal neurons.Here we report that enhancement of CICR by caffeine led to an increase in sIAHPamplitude, while inhibition of CICR by ryanodine caused a small, but significant reduction of sIAHP. Inhibition of ryanodine-sensitive Ca2+stores by ryanodine or depletion by the SERCA pump inhibitor cyclopiazonic acid caused a substantial attenuation in the sIAHPactivity-dependent potentiation in both rat and mouse CA1 pyramidal neurons. Neurons from mice lacking RyR3 receptors exhibited a sIAHPwith features undistinguishable from wild-type neurons, which was similarly reduced by ryanodine. However, the lack of RyR3 receptors led to a faster and reduced activity-dependent potentiation of sIAHP.We conclude that ryanodine receptor-mediated CICR contributes both to the amplitude of the sIAHPat steady state and its activity-dependent potentiation in rat and mouse hippocampal pyramidal neurons. In particular, we show that RyR3 receptors play an essential and specific role in shaping the activity-dependent potentiation of the sIAHP. The modulation of activity-dependent potentiation of sIAHPby RyR3-mediated CICR contributes to plasticity of intrinsic neuronal excitability and is likely to play a critical role in higher cognitive functions, such as learning and memory.

Carbon nanotubes (CNTs) are among the most exploited carbon allotropes in the emerging technologies of molecular sensing and bioengineering. However, the advancement of algal nanobiotechnology and nanobionics is hindered by the lack of methods for the straightforward visualization of the CNTs inside the cell. Herein, we present a handy and label-free experimental strategy based on visible Raman microscopy to assess the internalization of single-walled carbon nanotubes (SWCNTs) using the model photosynthetic alga Chlamydomonas reinhardtii as a recipient. The relationship between the properties of SWCNTs and their biological behavior was demonstrated, along with the occurrence of excitation energy transfer from the excited chlorophyll molecules to the SWCNTs. The non-radiative deactivation of the chlorophyll excitation promoted by the SWCNTs enables the recording of Raman signals originating from cellular compounds located near the nanotubes, such as carotenoids, polyphosphates, and starch. Furthermore, the outcome of this study unveils the possibility to exploit SWCNTs as spectroscopic probes in photosynthetic and non-photosynthetic systems where the fluorescence background hinders the acquisition of Raman scattering signals.

Mesenchymal stem cells (MSCs) are well-characterized adult stem cells, recently isolated from human nucleus pulposus of degenerate and non-degenerate intervertebral disc. The attention to this source is linked to its embryologic history and cells may conserve a stronger aptitude to neuronal differentiation than other MSCs. Here, MSCs from nucleus pulposus (NP-MSCs) were successfully isolated and characterized for morphology, proliferation, and expression of selected genes. Subsequently, the neuronal differentiation was induced by 10 days of culture with a neuronal medium. NP-MSCs subjected to neural differentiation media (NP-MSCs-N) showed a morphological and biochemical modifications. NP-MSCs-N displayed elongated shape with protrusion, intermediate filaments, microtubules, and electron dense granules and the ability to form neurospheres. Even if they expressed neural markers such as NESTIN, β-TUBULIN III, MAP-2, GAP-43, and ENOLASE-2, the neural differentiated cells did not show neither spontaneous nor evoked intracellular calcium variations compared to the undifferentiated cells, suggesting that cells do not have electric functional properties. Further studies are required in order to get a better understanding and characterization of NP-MSCs and analyzed the molecular mechanisms that regulate their neural differentiation potential.

During senescence, cells undergo distinctive biochemical and morphological changes and become dysfunctional. MiRNAs are involved in the senescence process and specific miRNAs can localize to mitochondria (mitomiRs). We hypothesized that part of the typical alterations of senescence may depends on mitomiRs deregulation. Therefore, we thoroughly explored the phenotype of human endothelial cells undergoing replicative senescence (sHUVECs) and observed elongated/branched mitochondria, accumulation of autophagic vacuoles (AVs), increased ROS and IL-1β production and reduced expression of Bcl-2 compared to younger cells (yHUVECs). Despite these pro-apoptotic features, sHUVECs are more resistant to serum deprivation, conceivably due to development of pro-survival strategies such as upregulation of Bcl-xL and Survivin. We demonstrate that mitomiR-181a, -34a, and -146a, are overexpressed and localize to mitochondria in sHUVECs compared with yHUVECs and that they: i) down-regulate Bcl-2, ii) induce permeability transition pore opening and activation of caspase-1 and 3, iii) affect sensitivity to apoptosis and iv) promote the conversion of LC3-I to LC3-II. Overall, we document for the first time that some mitomiRs can act as mediators of the multiple but functionally linked biochemical and morphological changes that characterize aging cells and that they can promote different cellular outcomes according to the senescence status of the cell.

Amyotrophic lateral sclerosis (ALS) causes rapidly progressive paralysis and death within 5 years from diagnosis due to degeneration of the motor circuits. However, a significant population of ALS patients also shows cognitive impairments and progressive hippocampal pathology. Likewise, the mutant SOD1(G93A) mouse model of ALS (mSOD1), in addition to loss of spinal motor neurons, displays altered spatial behavior and hippocampal abnormalities including loss of parvalbumin-positive interneurons (PVi) and enhanced long-term potentiation (LTP). However, the cellular and molecular mechanisms underlying these morpho-functional features are not well understood. Since removal of TrkB.T1, a receptor isoform of the brain-derived neurotrophic factor, can partially rescue the phenotype of the mSOD1 mice, here we tested whether removal of TrkB.T1 can normalize the number of PVi and the LTP in this model. Stereological analysis of hippocampal PVi in control, TrkB.T1−/−, mSOD1, and mSOD1 mice deficient for TrkB.T1 (mSOD1/T1−/−) showed that deletion of TrkB.T1 restored the number of PVi to physiological level in the mSOD1 hippocampus. The rescue of PVi neuron number is paralleled by a normalization of high-frequency stimulation-induced LTP in the pre-symptomatic mSOD1/T1−/− mice. Our experiments identified TrkB.T1 as a cellular player involved in the homeostasis of parvalbumin expressing interneurons and, in the context of murine ALS, show that TrkB.T1 is involved in the mechanism underlying structural and functional hippocampal degeneration. These findings have potential implications for hippocampal degeneration and cognitive impairments reported in ALS patients at early stages of the disease.

Brain-derived neurotrophic factor (BDNF) is a potent modulator of brain synaptic plasticity. Signaling defects caused by dysregulation of its Ntrk2 (TrkB) kinase (TrkB.FL) and truncated receptors (TrkB.T1) have been linked to the pathophysiology of several neurological and neurodegenerative disorders. We found that upregulation of Rbfox1, an RNA binding protein associated with intellectual disability, epilepsy and autism, increases selectively hippocampal TrkB.T1 isoform expression. Physiologically, increased Rbfox1 impairs BDNF-dependent LTP which can be rescued by genetically restoring TrkB.T1 levels. RNA-seq analysis of hippocampi with upregulation of Rbfox1 in conjunction with the specific increase of TrkB.T1 isoform expression also shows that the genes affected by Rbfox1 gain of function are surprisingly different from those influenced by Rbfox1 deletion. These findings not only identify TrkB as a major target of Rbfox1 pathophysiology but also suggest that gain or loss of function of Rbfox1 regulate different genetic landscapes.

The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication.

Epigallocatechin-3-gallate (EGCG) is a polyphenolic catechin from green tea, well known for being bioactive in age-associated pathologies where oxidative stress plays a preeminent role. The activity of this molecule is however contrasted by its high chemical and metabolic instability that determines a poor concentration of the antioxidant within the biological system after administration. In order to protect the molecule and increase its delivery efficiency, we have encapsulated EGCG inside anionic liposomes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesteryl hemisuccinate. To maximize EGCG internalization, magnesium salt was added in the preparation. However stable nanodispersions suitable for drug delivery were obtained only after treatment with Poloxamer-407, a polyethylene-propylene glycol copolymer. The structural and morphological properties of the produced dispersion were studied by X-ray diffraction, which showed a multilamellar structure even after EGCG addition and an ordering effect of Poloxamer-407; Dynamic Light Scattering demonstrated serum stability of the liposomes. The characterization was completed by evaluating both encapsulation efficiency (100%, in the final formulation) and in vitro EGCG release. Since oxidative stress is involved in numerous retinal degenerative diseases, such as age-related macular degeneration, the ability of these liposomes to contrast H2O2-induced cell death was assessed in human retinal cells. Morphological changes at the subcellular level were analyzed by Transmission Electron Microscopy, which showed that mitochondria were better preserved in cells treated with liposomes then those treated with free EGCG. In conclusion, the results demonstrated that the produced formulation enhances the efficacy of EGCG under stress conditions, thus representing a potential formulation for the intracellular delivery of EGCG in diseases caused by oxidative damage.

The integration of carbon nanotubes (CNTs) with plant machinery paved the way for various solutions to enhance plants vigor and their production efficiency [1,2]. Among others, the coupling of plant photosynthetic structures with nanomaterials has suggested the capability of single-walled CNTs (SWCNTs) to increase the efficiency of energy harnessing in the photosynthetic process [3]. This new knowledge may foster the exploitation of the nanotechnology tools to empower photosynthetic performance and production yields of commercially important microalgal species [4]. The research identified the physicochemical properties of SWCNTs enabling their entry into walled-cells of microalgae and developed experimental approaches to track their cell internalization. We tested the potential of the SWCNTs to improve algal fitness and photosynthetic performance by analysing the interaction of the carbon nanotubes and photochemical reactions in the model microorganism Chlamydomonas reinhardtii. The algal cultures were fed with stably dispersed SWCNTs and the effects of SWCNT-exposure on the function of Photosystem II and Photosystem I were studied under standard or stress-associated conditions. [1] Wang et al. 2016. Trends in Plant Science 21: 699-712. [2] Guatimosim et al. 2016. In: Bioengineering Applications of Carbon Nanostructures, A. Jorio (ed.), Springer Intern. Publ. Switzerland, pp. 17-29. [3] Giraldo et al. 2014. Nature Materials 13: 400-408. [4] Lambreva et al. 2015. Photosynthesis Research 125: 451-471.

BDNF exerts inotropic function in the adult mammalian heart through TrkB.T1 receptor and loss of this ligand/receptor system in cardiomyocytes impairs calcium signaling and causes cardiomyopathy, suggesting an essential physiological role for this pathway in cardiac function., Brain-derived neurotrophic factor (BDNF) is critical for mammalian development and plasticity of neuronal circuitries affecting memory, mood, anxiety, pain sensitivity, and energy homeostasis. Here we report a novel unexpected role of BDNF in regulating the cardiac contraction force independent of the nervous system innervation. This function is mediated by the truncated TrkB.T1 receptor expressed in cardiomyocytes. Loss of TrkB.T1 in these cells impairs calcium signaling and causes cardiomyopathy. TrkB.T1 is activated by BDNF produced by cardiomyocytes, suggesting an autocrine/paracrine loop. These findings unveil a novel signaling mechanism in the heart that is activated by BDNF and provide evidence for a global role of this neurotrophin in the homeostasis of the organism by signaling through different TrkB receptor isoforms.

Application of carbon nanotubes (CNTs) in plant biotechnology and agriculture brought to light an increasing amount of new findings, revealing the potential of CNTs to promote plant growth and crop production, or to control the delivery of fertilizers or pesticides to crops [1,2]. The coupling of plant cell structures with nanomaterials, used in the novel plant nanobionics approach, suggested the capability of CNTs to increase the efficiency of energy harnessing in the photosynthetic process and to improve cell response to oxidative stress conditions [3]. This new knowledge may foster the exploitation of the nanotechnology tools to empower photosynthetic performance and production yields of commercially important microalgal species. Large-scale cultivation of microalgae in photo-bioreactors plays an important role in the production of biomass, biofuels or high-value compounds [3]. One of the main problems in high-density-cultured algal bioreactors is the reduction of production yield due to occurrence of strong light-shading. Besides, very often, the introduction of different stress conditions (e.g. nutrients starvation) is exploited to redirect the algal metabolism towards the synthesis of desired compounds. Thus, experimental strategies taking advantages of CNT ability to assist the photosynthetic electron transport and the cell uptake of engineered nanoparticles or molecules with antioxidant or signaling functions bare great capacity to improve fitness and photosynthetic performance of commercially important photosynthetic microorganisms under large-scale manufacture conditions [4]. Here we will discuss the potential of the CNTs to enhance functions of algae facilitating a more efficient use of photosynthetic algal systems in the sustainable production of valuable goods. The research is focused on the development of experimental approaches to enable the interactions of photosynthetic microorganisms and CNTs, and to study the effects of CNT properties on algal fitness and photosynthetic performance, and nanotubes uptake into algal cell. [1] Wang et al., Trends Plant Sci. 21 (2016) 699-712. [2] Guatimosim et al., In: Bioengineering Applications of Carbon Nanostructures, A. Jorio (ed.), Springer Intern. Publ. Switzerland 2016, pp. 17-29. [3] Giraldo et al., Nature Mater. 13 (2014) 400-408. [4] Wong et al., Nature Mater. 16 (2017) 264-272. [5] Lambreva et al., Photosynth. Res. 125 (2015) 451-471.

Neurotrophin-dependent activation of the tyrosine kinase receptor trkB.FL modulates neuromuscular synapse maintenance and function; however, it is unclear what role the alternative splice variant, truncated trkB ( trkB.T1), may have in the peripheral neuromuscular axis. We examined this question in trkB.T1 null mice and demonstrate that in vivo neuromuscular performance and nerve-evoked muscle tension are significantly increased. In vitro assays indicated that the gain-in-function in trkB.T1−/−animals resulted specifically from an increased muscle contractility, and increased electrically evoked calcium release. In the trkB.T1 null muscle, we identified an increase in Akt activation in resting muscle as well as a significant increase in trkB.FL and Akt activation in response to contractile activity. On the basis of these findings, we conclude that the trkB signaling pathway might represent a novel target for intervention across diseases characterized by deficits in neuromuscular function.

Duchenne muscular dystrophy (DMD), a genetic disease arising from a mutation in the dystrophin gene, is characterized by muscle failure and is often associated with cognitive deficits. Studies of the dystrophic brain on the murine mdx model of DMD provide evidence of morphological and functional alterations in the central nervous system (CNS) possibly compatible with the cognitive impairment seen in DMD. However, while some of the alterations reported are a direct consequence of the absence of dystrophin, others seem to be associated only indirectly. In this review we reevaluate the literature in order to formulate a possible explanation for the cognitive impairments associated with DMD. We present a working hypothesis, demonstrated as an integrated neuronal network model, according to which within the cascade of events leading to cognitive impairments there are compensatory mechanisms aimed to maintain functional stability via perpetual adjustments of excitatory and inhibitory components. Such ongoing compensatory response creates continuous perturbations that disrupt neuronal functionality in terms of network efficiency. We have theorized that in this process acetylcholine and network oscillations play a central role. A better understating of these mechanisms could provide a useful diagnostic index of the disease's progression and, perhaps, the correct counterbalance of this process might help to prevent deterioration of the CNS in DMD. Furthermore, the involvement of compensatory mechanisms in the CNS could be extended beyond DMD and possibly help to clarify other physio-pathological processes of the CNS.

This paper explores the role of the calcium entry blocker nifedipine in the explanation of eccentric exercise-induced fibre damage by changes in skeletal muscle microcirculation. Eccentric exercise (EE) was induced by indirect stimulation of rat soleus muscle in its lengthening phase during cycling. Muscle damage was assessed by histology, electron microscopy and muscle tension 48 h later. Diameters of arterioles and venules, their response to dilator and constrictor stimuli and pattern of capillary flow were measured in epiiluminated muscles using intravital microscopy. Tetanic tension developed by EE muscles was lower (8.60 +/- 1.02, means +/- SEM, n = 8 N g(-1) wet weight compared to 12.25 +/- 0.56 in controls, P < 0.01). Electron microscopy showed changes similar to those in muscles exposed to EE by downhill running (Z line streaming, disruption of sarcolemma, swollen tubules). A total of 16% of muscle fibres were damaged, and fibre areas and interstitial space were enlarged. Capillary red blood cell flow showed tendency to a greater intermittency. Large venules were narrower, but arterioles and smaller venules had diameters similar to control muscles. Vessel dilatation to topically applied 10(-4) M adenosine was attenuated. Daily administration of calcium entry blocker nifedipine by gavage (2 mg/kg/day in two equal doses) removed the narrowing of venules, restored the dilator response of all vessels to adenosine and increased capillary:fibre ratio. The percentage of damaged fibres decreased to 4.7 and the size of the interstitial space and fibre areas was normalized. Thus muscle damage caused by eccentric exercise was attenuated by nifedipine due to its beneficial effect on muscle microcirculation, which was impaired by eccentric exercise.

Indirect chronic electrical stimulation of skeletal muscle activates not only efferent but also afferent nerve fibres. To investigate effects specific to this on capillary growth, one of the earliest changes, cell proliferation and capillary ultrastructure were studied in ankle flexors of rats with and without deafferentation of the stimulated side. Two weeks after preganglionic section of dorsal roots L4-L6, the peroneal nerve was stimulated (10 Hz, 8 h day(-1)) for 2 or 7 days. Proliferating nuclei labelled by bromodeoxyuridine or proliferating cell nuclear antigen staining were colocalized to alkaline phosphatase-stained capillaries (Lc) or other interstitial nuclei (Li) in frozen sections of extensor digitorum longus. Capillary fine structure was examined in extensor hallucis proprius by transmission electron microscopy. The stimulation-induced increase in capillary and interstitial proliferation (Lc 9.9 +/- 1.9 %, Li 8.8 +/- 2.1 % vs. Lc 2.6 +/- 0.4 %, Li 1.9 +/- 0.3 % in controls, P0.05) was depressed at 2 days by dorsal root section (Lc 4.8 +/- 0.7 %, Li 3.2 +/- 0.9 %, P0.05), an effect likely to be mainly on fibroblasts; no depression was seen at 7 days. Dorsal root section reduced stimulation-induced capillary endothelial swelling at both time points. In contralateral muscles of intact rats, stimulation increased interstitial cell proliferation and capillary swelling, both effects being eliminated by dorsal root section. Capillary growth induced by stimulation (24 % increase in capillary : fibre ratio at 7 days) was unaffected by deafferentation. The reduction in capillary ultrastructural changes and interstitial proliferation in both stimulated and contralateral muscles implies that stimulation of afferent fibres leads directly to release of humoral factors and/or activation via dorsal roots of fibres that release humoral substances. Contralateral muscles are an inadequate control for the effects of chronic stimulation in the intact animal.

MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case-control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54-62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes' IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.

Electron microscopy was used in order to study the change of the actin-containing filaments when the bound glycolytic enzymes were removed by a high ionic strength solution. The effectiveness of the extraction medium was checked by estimating the aldolase activity released. Evidence was provided that the larger diameter of the actin filaments in the I-bands in comparison with the A-bands can be accounted for by the bound glycolytic enzymes.

The control of peripheral structural plasticity of motor neurons by primary sensory neurons was studied in rat extensor digitorum longus (EDL) muscle. Polyinnervation of muscle fibers, sprouting and the motor neuron peripheral field size following L4 dorsal root cutting were evaluated using three different approaches: intracellular recording of end plate potentials, histochemical demonstration of sprouting and polyinnervation and in vivo recording of nerve-evoked twitch. Nodal sprouting was found in rhizotomized rats but not in controls and consistently muscle polyinnervation appeared. The muscle portion innervated by L3 ventral root was relatively reduced and that innervated by L5 was relatively enlarged: a trend to caudal shift of muscle innervation arose in rhizotomized rats. A control of motor neuron plasticity by primary sensory neurons is suggested.

We report the effects of a single gamma irradiation delivered to the soleus muscle of one limb of normal and mdx mice at the age of 16-20 days. At 45, 75 and 90 days of age transverse cryostat sections from the mid-belly of the muscles were used for microscopic examination. In normal mice the growth of fibres was appreciably reduced by irradiation without fibre loss. In the irradiated soleus of mdx mice the number of the regenerated centrally nucleated fibres was very small and the total number of fibres was remarkably reduced. The number of the peripherally nucleated fibres, presumably surviving since the birth of the animal, was almost consistently larger than in the contralateral non-irradiated limb. The cross-sectional area of the irradiated fibres was smaller. It is well known that proliferation and fusion of satellite cells are required both for regeneration after fibre damage and for the normal postnatal growth of muscle fibres: irradiation appears to reduce regeneration and growth. It is suggested that irradiation reduces damage by reducing fusion associated with growth. Our hypothesis indirectly indicates a significant link between dystrophin deficiency and fibre necrosis and accounts well for many features of mouse dystrophy under natural and experimental conditions.

We have used electron microscopy and computer image processing to produce a three-dimensional reconstruction of F-actin filaments decorated with the putative lectin and actin-binding protein comitin. These reconstructions show that comitin binds to F-actin at high radius primarily to actin subdomain 1. This location is distinctly different from the binding site on F-actin for other actin bundling proteins, such as members of the alpha-actinin family, and may result from the positively charged comitin interacting with negatively charged sites near the actin N terminus in subdomain 1. The location of the comitin binding site and its restriction to subdomain 1 on a single actin monomer is consistent with comitin's having a function distinct from other actin-binding proteins and, for example, would enable comitin to link bundled actin filaments to the Golgi.

The molecular mechanisms underlying the effects of electroconvulsive shock (ECS) therapy, a fast-acting and very effective antidepressant therapy, are poorly understood. Changes related to neuroplasticity, including enhanced adult hippocampal neurogenesis and neuronal arborization, are believed to play an important role in mediating the effects of ECS. Here we show a dynamic upregulation of the scaffold protein tamalin, selectively in the hippocampus of animals subjected to ECS. Interestingly, this gene upregulation is functionally significant because tamalin deletion in mice abrogated ECS-induced neurogenesis in the adult mouse hippocampus. Furthermore, loss of tamalin blunts mossy fiber sprouting and dendritic arborization caused by ECS. These data suggest an essential role for tamalin in ECS-induced adult neuroplasticity and provide new insight into the pathways that are involved in mediating ECS effects.

Duchenne muscular dystrophy (DMD) is characterized by variable alterations of the dystrophin gene and by muscle weakness and cognitive impairment. We postulated an association between cognitive impairment and architectural changes of the hippocampal GABAergic system. We investigated a major subpopulation of GABAergic neurons, the parvalbumin-immunopositive (PV-I) cells, in the dorsal hippocampus of the mdx mouse, an acknowledged model of DMD. PV-I neurons were quantified and their distribution was compared in CA1, CA2, CA3, and dentate gyrus in wild-type and mdx mice. The cell morphology and topography of PV-I neurons were maintained. Conversely, the number of PV-I neurons was significantly increased in the mdx mouse. The percent increase of PV-I neurons was from 45% for CA2, up to 125% for the dentate gyrus. In addition, the increased parvalbumin content in the mdx hippocampus was confirmed by Western blot. A change in the hippocampus processing abilities is the expected functional counterpart of the modification displayed by PV-I GABAergic neurons. Altered hippocampal functionality can be responsible for part of the cognitive impairment in DMD.

Pathological orin vitrooverexpression of the truncated TrkB (TrkB.T1) receptor inhibits signaling through the full-length TrkB (TrkB.FL) tyrosine kinase receptor. However, to date, the role of endogenous TrkB.T1 is still unknown. By studying mice lacking the truncated TrkB.T1 isoform but retaining normal spatiotemporal expression of TrkB.FL, we have analyzed TrkB.T1-specific physiological functions and its effect on endogenous TrkB kinase signalingin vivo. We found that TrkB.T1-deficient mice develop normally but show increased anxiety in association with morphological abnormalities in the length and complexity of neurites of neurons in the basolateral amygdala. However, no behavioral abnormalities were detected in hippocampal-dependent memory tasks, which correlated with lack of any obvious hippocampal morphological deficits or alterations in basal synaptic transmission and long-term potentiation.In vivoreduction of TrkB signaling by removal of one BDNF allele could be partially rescued by TrkB.T1 deletion, which was revealed by an amelioration of the enhanced aggression and weight gain associated with BDNF haploinsufficiency. Our results suggest that, at the physiological level, TrkB.T1 receptors are important regulators of TrkB.FL signalingin vivo. Moreover, TrkB.T1 selectively affects dendrite complexity of certain neuronal populations.

Duchenne muscular dystrophy (DMD), a genetic disease due to dystrophin gene mutation and characterised by skeletal muscle failure, is associated with non-progressive cognitive deficits. In human and mouse brain, full-length dystrophin is localised postsynaptically in neocortical, hippocampal and cerebellar neurons. Evidence obtained in the CNS of dystrophic mice (mdx) suggested alterations of the GABAergic system. However, a direct functional evaluation of GABAergic synaptic transmission in mdx mice has not been conducted in the hippocampus, which is involved in cognitive processes and is rich in full-length dystrophin. Here, we investigated evoked and miniature inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons of mdx mice with patch clamp recording techniques. Results showed an increased frequency of miniature spontaneous IPSCs in mdx mice compared with controls, whereas evoked IPSCs did not show significant variations. Paired-pulse facilitation (PPF) analysis showed lack of facilitation at short intervals in mdx mice compared with that in wild-type mice. Analysis of density of synapses that innervate CA1 pyramidal cell bodies did not indicate significant differences between mdx mice and controls. Therefore, we suggest that increased miniature spontaneous IPSC frequency is due to altered pre-synaptic release probability. The present findings are discussed in the light of the accrued evidence for alterations of inhibitory synaptic transmission in the brain of dystrophic mice.

Given the pivotal role of ion channels in neoplastic transformation, the aim of the present study has been to assess possible differences in the expression patterns of voltage-gated monovalent cationic (Na(+) and K(+)) currents between normal and neoplastic mesothelial cells (NM, MPM, respectively), and to evaluate the role of specific ion channels in mesothelioma cells proliferation, apoptosis, and motility. To achieve this aim, membrane currents expressed in NM and MPM cells derived from surgically-removed human specimens were investigated by means of patch-clamp electrophysiology. NM cells were found to express three main classes of K(+) currents, which were defined as K(IR), maxiK(Ca), and K(V) currents on the basis of their biophysical and pharmacological properties. Each of these K(+) currents was absent in MPM cells; by contrast, MPM cells revealed the novel appearance of tetrodotoxin (TTX)-sensitive voltage-gated Na(+) currents undetected in normal mesothelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR analysis of MPM cells transcripts showed significant expression of the mRNAs encoding for Na(V)1.2, and Na(V)1.6, and Na(V)1.7 (and less so for Na(V)1.3, Na(V)1.4, and Na(V)1.5) main voltage-gated sodium channel (VGSC) alpha-subunit(s). Interestingly, blockade of VGSCs with TTX decreased mesothelioma cell migration in in vitro motility assays; on the other hand, TTX failed to interfere with cell viability, proliferation, and apoptosis progression triggered by UV exposure. In summary, the results of the present study suggest that VGSCs expression in MPM cells may favor the increased motility of the neoplastic cells, a phenotypic feature often associated with the malignant phenotype.

Objective:This study explores whether the a1 blocker prazosin improves capillarisation, blood flow and muscle performance in chronically ischaemic muscles.Design:Rat skeletal muscles were made ischaemic by unilateral ligation of the common iliac artery. The animals received prazosin and muscle blood flow, performance and capillary supply were studied 2 or 5 weeks later.Materials and methods:Prazosin (50 mg/l) was given in drinking water to animals with limited or intact blood supply. Muscle isometric twitch tension and fatigue index and blood flow at rest and during contractions (estimated by radio labelled microspheres) were measured in tibialis anterior, TA, and extensor digitorum longus, EDL. Capillary supply (capillary/fibre ratio, C/F) was estimated in frozen muscle sections stained for alkaline phosphatase in TA, EDL and soleus, S.Results:Prazosin increased significantly capillary supply in ischaemic muscles after 2 and 5 weeks and blood flow during contractions (control muscles 163±6 ml. 100 g−1 min−1; ligated 2 weeks 25±7, ligated + prazosin 116±38; ligated 5 weeks 27±7, + prazosin 137±29, p

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into several cell types. Bone marrow (BM)-MSCs mainly differentiate into osteoblasts or adipocytes. MSC interactions with their microenvironment directly affect their self-renewal/differentiation program. Here, we show for the first time that Fas ligand (FasL), a well-explored proapoptotic cytokine, can promote proliferation of BM-derived MSCs in vitro and inhibits their differentiation into adipocytes. BM-MSCs treated with a low FasL dose (0.5 ng/ml) proliferated more rapidly than untreated cells without undergoing spontaneous differentiation or apoptosis, whereas higher doses (25 ng/ml) induced significant though not massive BM-MSC death, with surviving cells maintaining a stem cell phenotype. At the molecular level, 0.5 ng/ml FasL induced ERK1/2 phosphorylation and survivin upregulation, whereas 25 ng/ml FasL induced caspase activation. Importantly, 25 ng/ml FasL reversibly prevented BM-MSC differentiation into adipocytes by modulating peroxisome proliferator-activated receptor gamma (PPARγ) and FABP4/aP2 expression induced by adipogenic medium. All such effects were inhibited by anti-Fas neutralizing antibody. The in vitro data regarding adipogenesis were confirmed using Fas(lpr) mutant mice, where higher PPARγ and FABP4/aP2 mRNA and protein levels were documented in whole tibia. These data show for the first time that the FasL/Fas system can have a role in BM-MSC biology via regulation of both proliferation and adipogenesis, and may have clinical relevance because circulating Fas/FasL levels decline with age and several age-related conditions, including osteoporosis, are characterized by adipocyte accumulation in BM.

1. 1. Functional studies were performed to evaluate the effects of some muscarinic agents at the neuromuscular junction of the mouse. 2. 2. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analyzed by means of extracellular recording. The amplitude of spontaneous and of evoked acetylcholine release, the frequency of spontaneous acetylcholine release and the time course of the quantal release have been measured by means of an EPC7 patch clamp amplifier. 3. 3. This electrophysiological method revealed multiple dose-related effects of some agonists and antagonists on the above parameters. Concentration-response curves related to the parameters underlying the function of this cholinergic synapse were obtained and the apparent EC50 values calculated. 4. 4. Many of the interactions of the agonists and antagonists could inhibit neuromuscular transmission. The rank order potencies related to the inhibition of the evoked signals were carbachol > oxotremorine >d,l-muscarine for the agonists and methoctramine > 4-DAMP >l-hyoscyamine > AFDX-116 > ipratropium > pirenzepine for the antagonists. 5. 5. These findings suggest a more complicated pattern related to the muscarinic action at the mouse neuromuscular junction with the involvement of some post-synaptic located sites.

Functional studies were performed to evaluate the effects of methoctramine at the neuromuscular junction of the mouse. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analysed by means of the extracellular recording with an EPC7 Patch Clamp amplifier. This electrophysiological method revealed a dose-related inhibitory effect of methoctramine on the studied parameters. The dramatic reduction of the kinetics of the quantal conductance change indicates an action at the postsynaptic level. The effects of methoctramine have been compared with those of the muscarinic agonist oxotremorine. Concentration/response curves for the two drugs were obtained and the apparent EC50 values calculated. The effects of oxotremorine were not antagonized by 1 microM methoctramine. These findings suggest an interaction of some muscarinic agents on the postsynaptic receptor-ion-channel complex at the mouse neuromuscular junction.

Some techniques to block muscular nerve evoked contraction involve pharmacological approaches using synaptic blocking agents. Such methods interfere with normal synaptic transmission, and could introduce artifacts making difficult the experimental interpretation. The method based on the use of formamide pre-treatment should not interfere with synaptic physiology, indeed previous works suggest that the mechanism involved in block of muscle activity could depend on the decrease in specific postsynaptic membrane capacitance, and on the disruption of the morphology of the transverse tubule system. To prove this assumption we evaluated before and after formamide pre-treatment, some pre and postsynaptic parameters related to the spontaneous quantal release (MEPC). By means of the Loose patch clamp technique, we demonstrated, that formamide pre-treatment increases in an irreversible manner the frequency of spontaneous quantal release. Morphology of MEPC appear not modified by formamide pretreatment, which does not interfere with postsynaptic cholinergic receptors activity.

The present paper describes a simple HPLC method designed for measuring high-energy phosphate (HEP) compounds in a single run and inorganic phosphate (Pi) in an other short run under the same HPLC conditions. Inorganic phosphate was estimated by using thymidine phosphorylase (EC 2.4.2.4) which catalyzes a reaction involving inorganic phosphate to produce 2-deoxyribose 1-phosphate and thymine. The thymine/Pi stoichiometry was 1. The method provides a reproducible instrument for evaluating myocardial high-energy metabolism under physiological and pathological conditions.