Exercise as a new physiological stimulus for brown adipose tissue activity.

BACKGROUND AND AIM: Brown adipose tissue (BAT) plays a major role in body energy expenditure counteracting obesity and obesity-associated morbidities. BAT activity is sustained by the sympathetic nervous system (SNS). Since a massive activation of the SNS was described during physical activity, we investigated the effect of endurance running training on BAT of young rats to clarify the role of exercise training on the activity and recruitment state of brown cells. METHODS AND RESULTS: Male, 10-week-old Sprague Dawley rats were trained on a motor treadmill (approximately 60% of VO2max), 5 days/week, both for 1 and 6 weeks. The effect of endurance training was valuated using morphological and molecular approaches. Running training affected on the morphology, sympathetic tone and vascularization of BAT, independently of the duration of the stimulus. Functionally, the weak increase in the thermogenesis (no difference in UCP-1), the increased expression of PGC-1α and the membrane localization of MCT-1 suggest a new function of BAT. Visceral fat increased the expression of the FOXC2, 48 h after last training session and some clusters of UCP-1 paucilocular and multilocular adipocytes appeared. CONCLUSION: Exercise seemed a weakly effective stimulus for BAT thermogenesis, but surprisingly, without the supposed metabolically hypoactive effects. The observed browning of the visceral fat, by a supposed white-to-brown transdifferentiation phenomena suggested that exercise could be a new physiological stimulus to counteract obesity by an adrenergic-regulated brown recruitment of adipocytes.

Heart Rate Variability reveals the fight between racially biased and politically correct behaviour.

In this study, we explored vagally-mediated heart rate variability (vmHRV) responses, a psychophysiological index of cognitive self-regulatory control, to map the dynamics associated with empathic responses for pain towards an out-group member. Accordingly, Caucasian participants were asked to judge the experience of African and Caucasian actors touched with either a neutral or a harmful stimulus. Results showed that (1) explicit judgment of pain intensity in African actors yielded higher rating score and (2) took longer time compared to Caucasian actors, (3) these behavioural outcomes were associated with a significant increment of RMSSD, Log-HF-HRV and HF-HRV n.u., (4) resting HF-HRV n.u. predicted the participants' lag-time to judge painful stimulations delivered to African actors. Interestingly, these dynamics were associated with a measure of implicit racial attitudes and were, in part, abolished when participants performed a concurrent task during videos presentation. Taken together our results support the idea that a cognitive effort is needed to self-regulate our implicit attitude as predicted by the 'Contrasting Forces Model'.

Surface and inner cell behaviour along skeletal muscle cell in vitro differentiation.

During muscle tissue differentiation, in particular in the formation of myotubes from the myoblasts, plasma membrane changes its morpho-functional characteristics. In this study, muscle cell membrane behaviour has been studied along the differentiation of C2C12, a mouse myoblastic adherent cell line. Flat undifferentiated cells, cultured for 3-4 days in the differentiation medium, progressively become thick, long and multinucleated myotubes covered with microvilli. They lose stress fibers and adhesion to the underlying substrate evidentiating an actin redistribution, followed by the spatial organization of thick and thin myofilaments. Sarcomeres and myofibrils occasionally appear, even if a certain percentage of "myosacs" containing randomly oriented filaments can be identified all along the differentiation. M-cadherin, a molecule involved in cell-cell adhesion, also appears in the early differentiation stage, during myoblast fusion. Occasional focal contractions can also be observed in myotubes, which prompt an electrophysiological membrane analysis. When studied by means of patch clamp technique, resting membrane potential appears to undergo a transient depolarization, while input resistance increases until day 5 after differentiation induction, then successively decreases. Capacitance declines until day 5, later appearing enhanced. Moreover, with the induction of differentiation, the pattern of functional voltage-dependent ion channels changes. Therefore, during myogenesis, cell maturation is coupled with changes in cell membrane morphological features and functional characteristics.

Effects of nanosilver on Mytilus galloprovincialis hemocytes and early embryo development.

Silver nanoparticles (AgNP), one of the main nanomaterials for production and use, are expected to reach the aquatic environment, representing a potential threat to aquatic organisms. In this study, the effects of bare AgNPs (47 nm) on the marine mussel Mytilus galloprovincialis were evaluated at the cellular and whole organism level utilizing both immune cells (hemocytes) and developing embryos. The effects were compared with those of ionic Ag+(AgNO3). In vitro short-term exposure (30 min) of hemocytes to AgNPs induced small lysosomal membrane destabilization (LMS EC50 = 273.1 µg/mL) and did not affect other immune parameters (phagocytosis and ROS production). Responses were little affected by hemolymph serum (HS) as exposure medium in comparison to ASW. However, AgNPs significantly affected mitochondrial membrane potential and actin cytoskeleton at lower concentrations. AgNO3 showed much higher toxicity, with an EC50 = 1.23 µg/mL for LMS, decreased phagocytosis and induced mitochondrial and cytoskeletal damage at similar concentrations. Both AgNPs and AgNO3 significantly affected Mytilus embryo development, with EC50 = 23.7 and 1 µg/L, respectively. AgNPs caused malformations and developmental delay, but no mortality, whereas AgNO3 mainly induced shell malformations followed by developmental arrest or death. Overall, the results indicate little toxicity of AgNPs compared with AgNO3; moreover, the mechanisms of action of AgNP appeared to be distinct from those of Ag+. The results indicate little contribution of released Ag+ in our experimental conditions. These data provide a further insight into potential impact of AgNPs in marine invertebrates.

Bdnf expression in rat skeletal muscle after acute or repeated exercise.

Brain derived growth factor (BDNF) gene of rat has a complex structure: at least four 5' untranslated exons regulated by different promoters and one 3' exon containing the encoding region. BDNF is expressed by skeletal muscles in an activity-dependent manner. In this study, BDNF mRNA was analysed by RT-PCR in the soleus muscle following a single (acute) session of running or a training of five days of running (repetitive exercise). Moreover, the expression of the exons was quantitatively analysed by real time RT-PCR. Finally, muscle BDNF protein level was evaluated by western blotting. BDNF mRNA was found to increase over the second day after acute exercise; on the other hand, two peaks (2 and 24 hours after the last session, respectively) in BDNF mRNA level were found after repetitive exercise, but it was similar to that of controls 6 hours after the last session. BDNF protein level progressively increased also after the mRNA went back to the basal level, so suggesting that it cumulates within the cell after acute exercise, whereas it followed the mRNA level time course after repetitive exercise. These results point to the following conclusions: BDNF mRNA is up-regulated by activity, but this response is delayed to the second day after acute exercise; repetitive exercise transiently depresses the expression of BDNF mRNA, so that the over-expression due to the previous day's exercise completely disappears 6 hours after the last exercise session.

Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus.

The fate of adult-generated neurons in dentate gyrus is mainly determined early, before they receive synapses. In developing brain, classical neurotransmitters such as GABA and glutamate exert trophic effects before synaptogenesis. In order for this to occur in adult brain as well, immature non-contacted cells must express functional receptors to GABA and glutamate. In this investigation, patch-clamp recordings were used in adult rat dentate gyrus slices to assess the presence and analyze the characteristics of GABA- and glutamate-evoked currents in highly immature, synaptically-silent granule cells. Whole-cell patch-clamp recordings showed that all the analyzed cells responded to puff application of GABA and most of them responded to glutamate. Currents evoked by GABA were mediated exclusively by GABAA receptors and those elicited by glutamate were mediated by NMDA and AMPA/Kainate receptors. GABAA receptor-mediated currents were reduced by furosemide, which suggests that synaptically-silent immature neurons express high-affinity, alpha4-subunit-containing GABAA receptors. Gramicidin-perforated-patch recordings showed that GABAA receptor-mediated currents exerted a depolarizing effect due to high intracellular chloride concentration. Synaptically-silent immature cells shared morphological and electrophysiological properties with GFP-expressing, 7-day-old adult-generated granule layer cells, indicating that they could be in the first week of life, the period of maximal newborn cell death. Moreover, the presence of functional GABA and glutamate receptors was confirmed in these GFP-expressing cells. Present findings are mostly consistent with previous data obtained in female mice undergoing spontaneous activity and in transgenic mice, except for some inconsistencies about the presence of functional glutamatergic receptors. We speculate that adult-generated, non-contacted granule cells may be able to sense activity-related variations of GABA and glutamate extracellular levels. This condition is necessary, even if not sufficient, for these neurotransmitters to have a direct role in addressing cell survival.

Maternal creatine supplementation affects the morpho-functional development of hippocampal neurons in rat offspring.

Creatine supplementation has been shown to protect neurons from oxidative damage due to its antioxidant and ergogenic functions. These features have led to the hypothesis of creatine supplementation use during pregnancy as prophylactic treatment to prevent CNS damage, such as hypoxic-ischemic encephalopathy. Unfortunately, very little is known on the effects of creatine supplementation during neuron differentiation, while in vitro studies revealed an influence on neuron excitability, leaving the possibility of creatine supplementation during the CNS development an open question. Using a multiple approach, we studied the hippocampal neuron morphological and functional development in neonatal rats born by dams supplemented with 1% creatine in drinking water during pregnancy. CA1 pyramidal neurons of supplemented newborn rats showed enhanced dendritic tree development, increased LTP maintenance, larger evoked-synaptic responses, and higher intrinsic excitability in comparison to controls. Moreover, a faster repolarizing phase of action potential with the appearance of a hyperpolarization were recorded in neurons of the creatine-treated group. Consistently, CA1 neurons of creatine exposed pups exhibited a higher maximum firing frequency than controls. In summary, we found that creatine supplementation during pregnancy positively affects morphological and electrophysiological development of CA1 neurons in offspring rats, increasing neuronal excitability. Altogether, these findings emphasize the need to evaluate the benefits and the safety of maternal intake of creatine in humans.

Neurogenesis in the adult rat dentate gyrus is enhanced by vitamin E deficiency.

Neurogenesis occurs throughout adult life in rat dentate gyrus. Factors and mechanisms of adult neurogenesis regulation are not well known. Vitamin E deficiency has been found to deliver a neurogenetic potential in rat dorsal root ganglia. To determine whether the role of tocopherols in adult neurogenesis may be generalized to the central nervous system, changes in adult rat dentate gyrus neurogenesis were investigated in vitamin E deficiency. Neurogenesis was quantitatively studied by determination of the density of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells and by determination of the total number of cells in the granule cell layer. The BrdU-labeled cells were immunocytochemically characterized by demonstration of neuronal marker calbindin D28K. The following results were found: (1) the volume of the granule layer increased in controls from 1 to 5 months of age, mainly due to cell density decrease; (2) the volume increased by a similar amount in vitamin E-deficient rats, mainly because of an increase in cell number; (3) BrdU-positive cells were more numerous in vitamin E-deficient rats in comparison to age-matched controls; (4) the increase in proliferated cells was located in the hilus and in the plexiform layer. This study confirms that neurogenesis occurs within adult dentate gyrus and demonstrates that this process is enhanced in vitamin E deficiency. This finding indicates that vitamin E may be an exogenous factor regulating adult neurogenesis.

Developmental changes in innervation of rat extensor digitorum longus muscle.

Motor neurons and ventral root motor fibres innervating extensor digitorum longus (EDL) muscle in rats of 0.5-3.5 months of age were studied by HRP-retrograde labelling and in vivo muscle tension recording, respectively. EDL nucleus size increased with age but motor neurons number and size did not change. Twitch and tetanus tension increased with age proportionally to muscle mass. At 0.5 months of age, but not at subsequent ages, the muscle was incompletely innervated functionally. The contribution to EDL muscle innervation came from L3, L4 (pre-eminently), and L5 ventral roots. The number of motor fibres running in L3-L5 ventral roots and innervating EDL muscle increased from 41.7 +/- 2.2 (mean +/- S.E.M.) at 0.5 months to 68.6 +/- 1.9 at 3.5 months (P < 0.001). The greatest changes in m.w., in tension and in number of alpha nerve fibres innervating the muscle occurred from 0.5 to 1.5 months of age; afterwards changes occurred, but at a slower rate. HRP labels all neurons innervating EDL muscle, while tension recordings allow the counting of alpha nerve fibres (not gamma) running in ventral roots; these differences may account for the different results obtained.

Vitamin E affects quantitative age changes in lumbar motoneurons and in their peripheral projections.

Vitamin E deficiency was previously found to induce plastic changes in the number of primary sensory neurons and in motoneuron peripheral field projections. In this work, quantitative changes in motoneurons of lumbar segments, in nerve fibres constituting ventral roots and in innervating leg motor fibres were studied in normal and vitamin E deficient rats from 1 to 5 months of age. The number of lumbar motoneurons was found to decrease, while there were no changes in the number of ventral root fibres. An increase in the number of innervating leg motor fibres was observed during ageing in control rats; in vitamin E deficient rats the number of fibres in the ventral roots did not change, as occurred in controls, but the decrease in the number of motoneurons was smaller and the number of innervating leg motor fibres increased further in comparison to the controls. The findings are consistent with the idea that vitamin E deficiency causes a decrease in motoneuron death or, alternatively, that it induces some process partially compensating naturally occurring motoneuron death.

Enlargement of motoneuron peripheral field following partial denervation with or without dorsal rhizotomy.

In partially denervated skeletal muscle, spared motor fibres sprout, enlarging motor unit size. Neuritogenesis and sprouting are known to depend on the synaptic input to the neurons. This suggests that spared motoneuron reaction to partial muscle denervation might be controlled by primary sensory neurons which directly or indirectly project to motoneurons. In two groups of rats, different surgical procedures were carried out: partial denervation of the extensor digitorum longus muscle without or with homolateral dorsal rhizotomy. Spared motoneuron peripheral field was evaluated by nerve-evoked tension measures. Following partial muscle denervation, spared motoneurons enlarged their projection peripheral field five to six times, innervating most of the denervated portion of the muscle. When dorsal rhizotomy was carried out together with partial denervation, the enlargement of the motoneuron's peripheral field occurred later; however, the peripheral field size was the same or greater than that found in partially denervated muscles without dorsal rhizotomy in the long term. Excitatory postsynaptic potential recordings at neuromuscular junctions consistently showed that innervation of denervated muscle cells by spared motoneurons was impaired when the dorsal roots were cut. Finally, in both groups of operated rats an increase in motor unit number occurred early after surgery, anticipating a process normally occurring in the same age range. These findings are consistent with the idea that sensory input trans-synaptically controls motoneuron peripheral field size.

Rat motor neuron plasticity induced by dorsal rhizotomy.

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.

Are there proliferating neuronal precursors in adult rat dorsal root ganglia?

The origin of new neurons in dorsal root ganglia of adult rat was investigated using an experimental model in which postnatal neurogenesis naturally occurring is enhanced and restricted in a brief period of life. Possible mitotic origin of new neurons was investigated by means of 5-bromo-2-deoxyuridine, anti-NF 200 antibody was used to detect if proliferated cells showed a neuronal phenotype. The results suggest that postnatal neurogenesis in dorsal root ganglia could depend only in part on precursor proliferation and that normally new neurons derive from the late differentiation of postmitotic cells.

Alpha-tocopherol controls cell proliferation in the adult rat dentate gyrus.

The effect of alpha-tocopherol on cell proliferation and proliferated cell survival was investigated in the dentate gyrus of adult rats. Adult rats were supplemented with alpha-tocopherol, injected with 5-bromo-2'-deoxyuridine (BrdU), that is incorporated into DNA during the S-phase, and killed at different time after BrdU injection. The number of newborn cells decreased after alpha-tocopherol supplementation, confirming the hypothesis that alpha-tocopherol is able to depress cell proliferation in vivo. Most newborn cells die within few days; more newborn cells survive in alpha-tocopherol-treated rats, suggesting the hypothesis that alpha-tocopherol decreases cell death.

Spatial learning affects immature granule cell survival in adult rat dentate gyrus.

Neurogenesis occurs throughout life in mammalian dentate gyrus. The effect of learning on newborn cell survival was studied in rat. Rats were trained on a hippocampus-dependent spatial learning task by using Morris water maze. Neurogenesis was evaluated by 5-bromo-2'deoxyuridine administered before learning. Several newborn cells expressed the immature neuron marker TOAD-64. The main findings were as follows: (1) the survival of newborn cells was enhanced by learning at early stage of differentiation; (2) the newborn cells saved by learning were mainly located in the rostral part of external blade of granule cell layer and (3) there was a correlation between the actual individual learning and newborn cell survival.

Age-related changes in maturation of regenerated motor innervation.

The dependence of spontaneous transmitter release by regenerated nerve endings on age was studied in rat extensor digitorum longus muscle after sciatic nerve crush during the first year of life. Intracellular recordings were carried out at different times after denervation in muscles of 1.5-, 3- or 12-month-old animals. The frequency of miniature endplate potentials was detected. In order to determine the percentage of multiple innervation, nerve-evoked endplate potentials were recorded. The time course of the percentage of polyinnervated muscle cells was similar in the three groups of animals, but mepp frequency increased more slowly with increasing age. Moreover, by extrapolating the intercept of linear regression for mepp frequency vs days from denervation, a conventional initial time of reinnervation may be computed: the results are consistent with a decrease in the nerve regeneration rate during the first year of life.

Response of fast muscle innervation to hypothyroidism.

The early period of motor innervation development is characterized by multiple innervation of muscle cells. This transitory state in rat extensor digitorum longus (edl) muscle is normally concluded at weaning when a 1:1 ratio between nerve endings and muscle cells is reached. Motor innervation of edl muscle in rats made hypothyroid after weaning was studied in three ways: electrophysiology (intracellular recordings of muscle postsynaptic potentials) was carried out to study neuromuscular transmission; silver impregnation of terminal axons to observe sprouting; force production in twitch and tetanus following direct muscle stimulation and nerve stimulation. A number of multiply innervated muscle cells was found in hypothyroid rats following two months of treatment. This finding seems to be related to the appearance of nodal sprouting in motor axons. No sign of denervated end-plates was found. Twitch and tetanus tension were smaller than in controls, but they were bigger when referred to unitary muscle mass. Time course of twitch, particularly half relaxation, was slowed in muscles of hypothyroid rats. These findings suggest that plastic processes occur in muscle innervation of rats made hypothyroid after weaning. Therefore, thyroid hormones play a role in stabilizing motor innervation not only during development, but also in adults.

Nodal and terminal sprouting by regenerating nerve in vitamin E-deficient rats.

The increased number of poly-innervated cells in normal and reinnervated extensor digitorum longus (edl) muscle of vitamin E-deficient rats suggests enhanced sprouting by motor neurons in conditions of decreased protection against lipid peroxidation. End-plates and terminal axons were observed by a combined technique that shows both end-plate acetylcholinesterase area and axons. Quantitative observations of nodal and terminal sprouting in normally innervated and reinnervated edl muscles of vitamin E-deficient rats were carried out. Branch points of nerve terminal within end-plates were also observed. Three main results were obtained. First, a notable increase of both terminal and nodal sprouting was found in reinnervated muscles of normal and vitamin E-deficient rats; moreover, a relative increase in the number of nodal sprouts occurs in the long run. Second, in muscles of uninjured, vitamin E-deficient rats, nodal and terminal sprouting and branching within end-plate was greater than in controls. Third, nodal sprouting by regenerating axons was more affected by vitamin E-deficiency than terminal sprouting and branching within end-plates.

Effect of ethanol on the maturation of the spontaneous transmitter release by regenerated nerve endings.

The cellular mechanisms involved in the theratogentic action of ethanol are not well known, but neuron outgrowth and synaptogenesis are regarded as the periods in which ethanol causes its major damage in the nervous system. The effects of chronic treatment with ethanol on the maturation of the spontaneous transmitter release by regenerated nerve endings in the rat were studied. The sciatic nerve was crushed and miniature end plate potentials (mepps) were recorded intracellularly in the re-innervated extensor digitorum longus muscle at different points in time after denervation; end plate potentials (epps) were also recorded. Two main effects were observed in the re-innervated muscles of ethanol-treated rats: (1) the appearance of spontaneous and evoked transmitter release was delayed and (2) the subsequent increase in frequency of mepps is faster. The possible mechanisms involved in these effects are discussed.

Effect of ethanol on the spontaneous transmitter release by nerve endings in the process of maturation.

Ethanol stimulates the spontaneous transmitter release from motor nerve endings, as shown by the increase of miniature end plate potential (m.e.p.p.) frequency at the neuro-muscular junction. The stimulation of acetylcholine spontaneous quantal release by ethanol is greater in regenerating than in mature nerve endings. The different effects of ethanol on regenerating nerve endings may be related to changes of chemical-physical membrane properties.