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Developing physical fitness (PF) behaviors early in life enhances physical development and facilitates sustained participation in physical activity and sports across childhood. This study addressed the effect of different teaching approaches on precursors of PF in kindergarten children. A total of 178 children (5.45 ± 0.40 years, female = 92) from 11 classes were organized into three groups. Group 1 (structured activity + free play) and Group 2 (only free play) attended the same playground (PrimoSport0246) for one hour/week for 10 weeks. Group 3 (structured activity + free play in kindergarten) followed their standard physical education curriculum at school. PF tests (standing long jump, medicine ball throw, and 20 m running speed) were administered pre- and post-intervention. Factorial ANOVA was implemented using a percentage change in PF performance (PFC) as the dependent variable, and teaching approaches, gender, and age as factors. Group 1 demonstrated significant improvements in fitness performance compared with Groups 2 and 3. Moderate to large effect sizes (Cohen’s d range: 0.68–1.40) were noted in both males and females. Six-year-old demonstrated the greatest improvement in composite PFC compared to Groups 2 and 3. A structured teaching plan, even with a limited dose of once per week, supported the development of PF attributes in kindergarteners.

Emerging evidence highlights the several roles that meninges play in relevant brain functions as they are a protective membrane for the brain, produce and release several trophic factors important for neural cell migration and survival, control cerebrospinal fluid dynamics, and embrace numerous immune interactions affecting neural parenchymal functions. Furthermore, different groups have identified subsets of neural progenitors residing in the meninges during development and in the adulthood in different mammalian species, including humans. Interestingly, these immature neural cells are able to migrate from the meninges to the neural parenchyma and differentiate into functional cortical neurons or oligodendrocytes. Immature neural cells residing in the meninges promptly react to brain disease. Injury-induced expansion and migration of meningeal neural progenitors have been observed following experimental demyelination, traumatic spinal cord and brain injury, amygdala lesion, stroke, and progressive ataxia. In this review, we summarize data on the function of meninges as stem cell niche and on the presence of immature neural cells in the meninges, and discuss their roles in brain health and disease. Furthermore, we consider the potential exploitation of meningeal neural progenitors for the regenerative medicine to treat neurological disorders.

Both the indoor and the outdoor environments and their organization exert pronounced influence upon physical activity behavior and motor development of preschool children. The aim of this study was to explore whether partly structured activity or free play in a specific playground had different impacts on motor competence development in 4–6-year-old preschoolers. The study had a pretest–post-test design, with two intervention groups and one control. Sixty-two children were included in a partly structured activity group and forty-three children in a free-play group. Both groups participated in playground activities consisting of 10 sessions (once a week), each lasting 1 h, in a specific playground setting. For the partly structured activity group, activities in each session consisted of a combination of both structured and free activity while the free-play group were engaged in unstructured play only. The control group did not attend the playground activities (N = 36). To assess levels of motor skills, each child completed pre- and post-tests using the Movement Assessment Battery for children (MABC-2), the Test of Motor Competence (TMC) and two playground-specific tests. A 3 (study group) and X 2 (gender) ANCOVAs were conducted on post-test scores on each of the test items from TMC, MABC-II and playground tests, with pre-test scores as covariates. Post hoc pairwise multiple comparisons were conducted with the alpha Bonferroni corrected, and the partial eta-squared (η2p) was applied as a measure of effect size. The results indicate no significant differences in motor competence measured by the TMC or the MABC-2 between groups. On the contrary, a significant improvement in performance in the playground-specific tests was observed in the partly structured activity group compared to the free-play and control groups.

Summary Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which can enable disease modeling and drug screening. However, their use for standardized large-scale drug screening studies is limited by their high batch-to-batch variability, long differentiation time (10–20 weeks), and high production costs. This is particularly relevant when brain organoids are obtained from human induced pluripotent stem cells (iPSCs). Here, we developed, for the first time, a highly standardized, reproducible, and fast (5 weeks) murine brain organoid model starting from embryonic neural stem cells. We obtained brain organoids, which progressively differentiated and self-organized into 3D networks of functional neurons with dorsal forebrain phenotype. Furthermore, by adding the morphogen WNT3a, we generated brain organoids with specific hippocampal region identity. Overall, our results showed the establishment of a fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modeling., Graphical abstract, Highlights • Murine embryonic NSCs are able to self-organize into brain organoids • Murine brain organoids mature in 32 days, showing dorsal forebrain identity • Brain organoids develop 3D network of functional neurons • WNT3a supplementation induces specific hippocampal brain region identity, Biological sciences; Neuroscience; Cell biology; Developmental biology

The link between motor and executive function (EF) development is unclear. To test whether learning difficult motor task drives EF development, we measured EF changes in 5-year-old children from 3 kindergartens in north-eastern Italy engaged in a specific motor program including a difficult motor task (walking on unstable bar). One-hundred children divided in 2 groups participated for 1 hour/week for 10 weeks: Group A was supported by a teacher and group B no. The capacity to perform the difficult task was assessed at the beginning and end of the study; the children able (Groups A1 and B1) or unable (Groups A2 and B2) to perform the task at the Pre-test were similar in the 2 groups. To assess EF, the day/night test (Gerstadt et al., 1994) was used. Dynamic balance results. Time at spring bar: Group A pre 69.2 post 29.6 - Group B pre 71.8 post 48.8. EF results. Group A1 pre 4.90 post 2.71 - Group B1 pre 3.62 post 3.54. Group A2 pre 4.64 post 1.22 - Group B2 pre 5.30 post 3.55. Conclusions. Teacher support significantly promoted improvement of motor skill; teacher’s scaffolding significantly promoted EF development in children with initial low motor competence only. This research was supported by Comitato Olimpico Nazionale Italiano CONI-Treviso and A.S.D. Laboratorio 0246, Treviso, Italy.

Research shows that the pandemic has increased sedentariness and decreased physical activity even in pre-schoolers. To deal with this period, the association Laboratorio 0246 proposed to some Italian kindergarten teachers an active training based on “ movement-stories ”. 39 teachers of kindergartens in 6 Italian regions were involved. All teachers completed the course a questionnaire, reporting difficulties related to space, time, bureaucratic demands, and highlighting problems in socializing with children due to restrictions. Most teachers reported that activities with storytelling in motion appeal to children, can be implemented in any indoor or outdoor setting, are easy to implement, and promote active children. There is a need for teacher training, including technical training about physical activity. Without the specific knowledge base, it is difficult for teachers to understand the real needs related to physical activity and to use methodology and didactics. The research-training-action pathway demonstrates to be well suited to address this situation.

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which promise to become relevant tools for disease modelling and drug screening. High-throughput drug screening assess a large amount of compounds using reliable and fast procedures. Organoids obtained from human induced pluripotent stem cells have high batch-to-batch variability, long differentiation time (10-20 weeks), and high cost required for production, thus limiting their use for standardized large-scale drug screening studies. Here, we developed, for the first time, a highly standardized, reproducible and fast (5 weeks) murine brain organoid model starting from multipotent neural stem cells (NSCs) isolated from embryonic subgranular zone (SGZ). Global transcriptome, protein expression, and cell metabolism data show that murine brain organoids progressively differentiate trough defined early, intermediate, and mature stages. During differentiation, brain organoids acquire a self-organized neural structure with a compact and interconnected neuronal layer at its edge. We measured neuronal activity on whole-brain organoids by combining electrophysiological techniques (patch-clamp and MEA recordings), transsynaptic tracing and calcium imaging analysis, and we found that, in less than 5 weeks of in vitro culture, these organoids own functional neurons organized in 3D networks with synaptic connections. Furthermore, we validated the potential of murine brain organoids to acquire a specific brain-region signature; by adding the morphogen WNT3a, we were able to generate brain organoids enriched in neurons with CA3 hippocampal region signature. Overall, our results showed the establishment of a novel fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modelling.

Dietary supplementation of essential amino acids (EAAs) has been shown to promote healthspan. EAAs regulate, in fact, glucose and lipid metabolism and energy balance, increase mitochondrial biogenesis, and maintain immune homeostasis. Basic science and epidemiological results indicate that dietary macronutrient composition affects healthspan through multiple and integrated mechanisms, and their effects are closely related to the metabolic status to which they act. In particular, EAA supplementation can trigger different and even opposite effects depending on the catabolic and anabolic states of the organisms. Among others, gut-associated microbial communities (referred to as gut microbiota) emerged as a major regulator of the host metabolism. Diet and host health influence gut microbiota, and composition of gut microbiota, in turn, controls many aspects of host health, including nutrient metabolism, resistance to infection, and immune signals. Altered communication between the innate immune system and the gut microbiota might contribute to complex diseases. Furthermore, gut microbiota and its impact to host health change largely during different life phases such as lactation, weaning, and aging. Here we will review the accumulating body of knowledge on the impact of dietary EAA supplementation on the host metabolic health and healthspan from a holistic perspective. Moreover, we will focus on the current efforts to establish causal relationships among dietary EAAs, gut microbiota, and health during human development.

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.

Engagement in difficult motor tasks promotes development of executive functions (EFs) (Diamond, 2020; Davis et al., 2011). In this study we analysed the effects of different types of scaffold on children exposed to a difficult motor task. Methodology. One hundred, 5 years old children from 2 kindergartens were engaged in a specific motor program for 1 hour/week for 10 weeks. The program included learning a difficult motor task (walking on an unstable bar) for which the instructor provided physical or physical + emotional scaffold. In addition, children were divided in two groups based on their initial level of capacity to perform the task. Levels of EFs were measured by the Day & Night test at the beginning and at the end of the training period. Data were analysed by two-way ANOVA. Results. The data suggest that in expert children the emotional support fosters the effect of the difficult task on the performance of the Day & Night test; while in beginners the emotional support counteracts the effects of physical scaffold resulting in a null effect on EF development. Conclusions. The data point to specificity of scaffold procedures for combining motor and cognitive development. This research was supported by Comitato Olimpico Nazionale Italiano CONI-Treviso and A.S.D. Laboratorio 0246, Treviso, Italy.

Ketamine is a drug of abuse with a unique profile, which besides its inherent mechanism of action as a non-competitive antagonist of the NMDA glutamate receptor, displays both antidepressant and reinforcing properties. The major aim of our study was to find a molecular signature of ketamine that may help in discriminating between its reinforcing and antidepressant effects. To this end, we focused our attention on BDNF, a neurotrophin that has been shown to play a role in both antidepressant and reinforcing properties of several drugs. Rats were exposed to self-administer intravenous (IV) ketamine (S/A) for 43 days or to receive a single IV ketamine 0.5mg/kg, or vehicle infusion. Although the dose we employed is lower than that reported by the literature, it however yields Cmax values that correspond to those achieved in humans after antidepressant treatment. Our results show that while the single infusion of ketamine increased the neurotrophin expression in the hippocampus while reducing it in the ventral striatum, a feature shared with other antidepressants, the repeated self-administration reduced mBDNF expression and its downstream signalling in both ventral striatum and hippocampus. Further, we here show that phosphorylation of Akt is oppositely regulated by ketamine, pointing to this pathway as central to the different actions of the drug. Taken together, we here point to BDNF and its downstream signalling pathway as a finely tuned mechanism whose modulation might subserve the different features of ketamine.

Motor development is affected by maturation and growth but also influenced by the specific environmental and cultural context. Therefore, cross-cultural research can provide information about how different cultural contexts, lifestyles, and physical activity contexts can influence the process of developing motor competence. The purpose of this study was to evaluate aspects of motor competence among children from different cultural backgrounds. The sample of 463 children from 6 to 8 years consisting of 132 Greek children (52.3 % boys), 126 Italian children (53.9 % boys), and 205 Norwegian children (52.7) completed the Test of Motor Competence (TMC) including two fine motor tasks-Placing Bricks-and Building Bricks and two gross motor tasks-Heel to Toe Walking and Walking/Running in Slopes. The results indicate that the Norwegian children performed better in all tests; the differences were statistically significant in all four tasks compared with Italian children and in two tasks compared with the Greek children (Building Bricks and Heel to Toe Walking). Greek children performed significantly better than the Italians in two tasks: Placing Bricks and Heel to Toe Walking. Italian children were significantly faster than the Greek ones in one task: Walking/Running in Slopes. The differences in terms of levels of basic fine and gross motor skills between children from the different countries may be a consequence of both different physical activity contexts and cultural policies, attitudes, and habits toward movement.

Mens sana in corpore sano. This quotation from the Roman poet Juvenal summarizes the common belief that a bond between physical health and cognition exists. The interest in the relation between physical activity and cognition started to grow when scientists first examined motor development. Development is a continuous phenomenon that occurs mostly during the early years of human life. The 'maturational perspective' of development dominated the first half of the 20th century; development was considered an internal or innate process guided by a biological and genetic clock controlling maturation of the central nervous system and development of the musculoskeletal system. Executive functions are a group of interrelated top-down mental processes responsible for selection, scheduling, coordination and monitoring of goal-directed processes regulating perception, memory and action. The practice of physical activity is believed to lead to improvement of motor skills and competencies with several beneficial effects on present and future health, as well as on psychological constraints of the child.

Oligodendrocyte loss can lead to cognitive and motor deficits. Current remyelinating therapeutic strategies imply either modulation of endogenous oligodendrocyte precursors or transplantation of in vitro expanded oligodendrocytes. Cell therapy, however, still lacks identification of an adequate source of oligodendrocyte present in adulthood and able to efficiently produce transplantable cells. Recently, a neural stem cell-like population has been identified in meninges. We developed a protocol to obtain high yield of oligodendrocyte lineage cells from one single biopsy of adult rat meningeal tissue. From 1 cm2 of adult rat spinal cord meninges, we efficiently expanded a homogenous culture of 10 millions of meningeal-derived oligodendrocyte lineage cells in a short period of time (approximately 4 weeks). Meningeal-derived oligodendrocyte lineage cells show typical mature oligodendrocyte morphology and express specific oligodendrocyte markers, such as galactosylceramidase and myelin basic protein. Moreover, when transplanted in a chemically demyelinated spinal cord model, meningeal-derived oligodendrocyte lineage cells display in vivo-remyelinating potential. This oligodendrocyte lineage cell population derives from an accessible and adult source, being therefore a promising candidate for autologous cell therapy of demyelinating diseases. In addition, the described method to differentiate meningeal-derived neural stem cells into oligodendrocyte lineage cells may represent a valid in vitro model to dissect oligodendrocyte differentiation and to screen for drugs capable to promote oligodendrocyte regeneration.

Clinical data with 3,4-methylenedioxymethamphetamine (MDMA) in post-traumatic stress disorder (PTSD) patients recently stimulated interest on the potential therapeutic use of psychedelics in disorders characterized by maladaptive memories, including substance use disorders (SUD). The rationale for the use of MDMA in PTSD and SUD is being extended to a broader beneficial “psychedelic effect,” which is supporting further clinical investigations, in spite of the lack of mechanistic hypothesis. Considering that the retrieval of emotional memories reactivates specific brain mechanisms vulnerable to inhibition, interference, or strengthening (i.e., the reconsolidation process), it was proposed that the ability to retrieve and change these maladaptive memories might be a novel intervention for PTSD and SUD. The mechanisms underlying MDMA effects indicate memory reconsolidation modulation as a hypothetical process underlying its efficacy. Mechanistic and clinical studies with other two classes of psychedelic substances, namely cannabinoids and ketamine, are providing data in support of a potential use in PTSD and SUD based on the modulation of traumatic and appetitive memory reconsolidation, respectively. Here, we review preclinical and clinical data on cannabinoids and ketamine effects on biobehavioral processes related to the reconsolidation of maladaptive memories. We report the findings supporting (or not) the working hypothesis linking the potential therapeutic effect of these substances to the underlying reconsolidation process. We also proposed possible approaches for testing the use of these two classes of drugs within the current paradigm of reconsolidation memory inhibition. Metaplasticity may be the process in common between cannabinoids and ketamine/ketamine-like substance effects on the mediation and potential manipulation of maladaptive memories.

Background: Sudden cardiac arrest is a major global health concern, and survival of patients with ischemia&ndash, reperfusion injury is a leading cause of myocardial dysfunction. The mechanism of this phenomenon is not well understood because of the complex pathophysiological nature of the disease. Aim of the study was to investigate the cardioprotective role of fingolimod in an in vivo model of cardiac arrest and resuscitation. Methods: In this study, an in vivo rat model of cardiac arrest using extracorporeal membrane oxygenation resuscitation monitored by invasive hemodynamic measurement was developed. At the beginning of extracorporeal life support (ECLS), animals were randomly treated with fingolimod (Group A, n = 30) or saline (Group B, n = 30). Half of the animals in each group (Group A1 and B1, n = 15 each) were sacrificed after 1 h, and the remaining animals (Group A2 and B2) after 24 h of reperfusion. Blood and myocardial tissues were collected for analysis of cardiac features, inflammatory biomarkers, and cell signaling pathways. Results: Treatment with fingolimod resulted in activation of survival pathways resulting into reduced inflammation, myocardial oxidative stress and apoptosis of cardiomyocytes. This led to significant improvement in systolic and diastolic functions of the left ventricle and improved contractility index. Conclusions: Sphingosine1phosphate receptor activation with fingolimod improved cardiac function after cardiac arrest supported with ECLS. Present study findings strongly support a cardioprotective role of fingolimod through sphingosine-1-phosphate receptor activation during reperfusion after circulatory arrest.

“Are new neurons added in the adult mammalian brain?” “Do neural stem cells activate following CNS diseases?” “How can we modulate their activation to promote recovery?” Recent findings in the field provide novel insights for addressing these questions from a new perspective. In this review, we will summarize the current knowledge about adult neurogenesis and neural stem cell niches in healthy and pathological conditions. We will first overview the milestones that have led to the discovery of the classical ventricular and hippocampal neural stem cell niches. In adult brain, new neurons originate from proliferating neural precursors located in the subventricular zone of the lateral ventricles and in the subgranular zone of the hippocampus. However, recent findings suggest that new neuronal cells can be added to the adult brain by direct differentiation ( e.g., without cell proliferation) from either quiescent neural precursors or non-neuronal cells undergoing conversion or reprogramming to neuronal fate. Accordingly, in this review we will also address critical aspects of the newly described mechanisms of quiescence and direct conversion as well as the more canonical activation of the neurogenic niches and neuroblast reservoirs in pathological conditions. Finally, we will outline the critical elements involved in neural progenitor proliferation, neuroblast migration and differentiation and discuss their potential as targets for the development of novel therapeutic drugs for neurodegenerative diseases.

Presence of neural stem cells in adult mammalian brains, including human, has been clearly demonstrated by several studies. The functional significance of adult neurogenesis is slowly emerging as new data indicate the sensitivity of this event to several "every day" external stimuli such as physical activity, learning, enriched environment, aging, stress and drugs. In addition, neurogenesis appears to be instrumental for task performance involving complex cognitive functions. Despite the growing body of evidence on the functional significance of NSC and despite the bulk of data concerning the molecular and cellular properties of NSCs and their niches, several critical questions are still open. In this work we review the literature describing i) old and new sites where NSC niche have been found in the CNS; ii) the intrinsic factors regulating the NSC potential; iii) the extrinsic factors that form the niche microenvironment. Moreover, we analyse NSC niche activation in iv) physiological and v) pathological conditions. Given the not static nature of NSCs that continuously change phenotype in response to environmental clues, a unique "identity card" for NSC identification is still lacking. Moreover, the multiple location of NSC niches that increase in diseases, leaves open the question of whether and how these structures communicate throughout long distance. We propose a model where all the NSC niches in the CNS may be connected in a functional network using the threads of the meningeal net as tracks.

The type 3 small conductance calcium-activated potassium channel (SK3) is expressed in embryonic and adult denervated skeletal muscles where it contributes to hyperexcitability. This study aimed at determining the role of muscle activity in regulating SK3 channels. Soleus muscles of adult rats were denervated by cutting the sciatic nerve. In reinnervation studies, the soleus nerve was crushed: in one group, muscles were reinnervated with electrically silent axons, by chronic sciatic nerve perfusion with tetrodotoxin. Several groups of denervated muscles were subjected to chronic direct electrical stimulation, using either fast (100 Hz) or slower patterns (20 or 30 Hz). The SK3 mRNA and protein levels in soleus muscle were determined by reverse transcriptional-PCR, Western blot and immunofluorescence. Both denervated and reinnervated–paralysed soleus muscles displayed similar up-regulation of SK3 mRNA and protein. Reinnervation with electrically active axons instead inhibited SK3 up-regulation. Chronic muscle direct stimulation in vivo, irrespective of the pattern used, reversed the denervation-induced up-regulation of SK3 expression or prevented it when initiated at the time of denervation. Chronic electrical stimulation of denervated muscles also completely prevented the development of the after-hyperpolarization (AHP) following the action potential, normally induced in the muscle fibres by denervation. We conclude that action potential activity evoked by motor neurones in muscle fibres is both necessary and sufficient to account for the physiological down-regulation of SK3 channels in the non-junctional membrane of skeletal muscle.

In this work, we define a GFP-tagged version of the p75 neurotrophin receptor (p75GFP) as a useful molecular tool for studying its distribution and cellular dynamics. Expression and subcellular localization of p75GFP have been characterized in non-neuronal (HEK 293) and in neuronal (cortical and hippocampal) cells. By monitoring movements of intracellular p75GFP in living cultured hippocampal neurons, we found that the chimeric protein was transported by tubulo-vesicular structures both anterogradely (0.1–0.5 μm/s) and retrogradely (0.1–1.1 μm/s), with a faster component in retrogradely moving structures. Movements of the p75GFP-containing structures were inhibited by treatment with the microtubule-disrupting agent nocodazole. Our data indicate that p75GFP is a reliable tool for studying spatial and cellular properties of p75 in CNS neurons and that p75 transport inside neurons is mediated by microtubule-associated motors.

The musculoskeletal system can be a target organ for adverse drug reactions (ADRs). Drug-induced muscle, bone or connective tissue injuries may be due to, i), primary direct drug action, or, ii), undirected consequence of generalized drug-induced disease. Musculoskeletal ADRs may be only temporarily disabling, such as muscle cramps, as well as in other cases may be serious and life-threatening, such as rhabdomyolysis. In the last few years there has been an increasing awareness of musculoskeletal ADRs. Some recent drug safety issues dealt with serious or uncommon musculoskeletal reactions like rhabdomyolysis associated to statins and tendon rupture associated to fluoroquinolones. In this review, we firstly selected those drug classes having a significantly high percentage of musculoskeletal disorder reports in the WHO adverse drug reaction database, maintained by the Uppsala Monitoring Centre. Secondly, the different musculoskeletal ADRs were closely analyzed through the data obtained from an Italian interregional ADRs spontaneous reporting database. The findings on drugs associated to different musculoskeletal disorders, have been integrated with a review of the epidemiological data available in the literature. For the most involved drugs (HMG-CoA reductase inhibitors, fluoroquinolones, corticosteroids, bisphosphonates, retinoids) the underlying musculoskeletal ADR mechanisms were also reviewed and discussed.

Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined. In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: (i) are a highly proliferating tissue; (ii) host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and (iii) are enriched in extracellular matrix components (e.g., fractones) known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood.

The regulative steps that control trafficking of ion channels are fundamental determinants of their qualitative and quantitative expression on the cell membrane. In this work the trafficking of the small conductance calcium-activated potassium channel, SK3 was studied in neurons in order to identify relevant molecular domains involved in this process. Hippocampal cell cultures were transfected with fusion proteins of green fluorescent protein (GFP) and different SK3 subunit truncations. The differential distribution of the mutants was analyzed by confocal microscopy and compared to the localization of the control fusion protein with full length SK3. The transport of chimeric proteins was quantified from fluorescence images by developing a morphometric analytical method. We found that the full length SK3 was distributed in cell body, axon and dendrites, whereas the deleted forms GFPΔ578–736 (deletion of the entire C-terminal domain), GFPΔCaMBD (deletion of the calmodulin-binding site) and GFPΔN (deletion of the N-terminal domain) were not transported into cell processes but accumulated in the cell body. The GFPΔ640–736 (deletion of the distal C-terminal domain) showed a distribution similar to control. The quantification and statistical analysis confirmed the differences in distribution across the three groups. In conclusion, the current work provides evidence for a fundamental role of the N-terminal domain and the calmodulin binding domain in SK3 trafficking in neurons.

Although cisplatin is effective in the treatment of different types of tumors, resistance to treatment is a major limitation. In an attempt of overcoming resistance mechanisms, a large effort has been made to generate compounds with a different geometry. At present, the most clinically relevant compounds include mononuclear (i.e. oxaliplatin) as well as multinuclear platinum complexes (i.e. BBR 3464). The mechanisms of cellular response to platinum complexes have not been completely elucidated. Among the main pathways affecting cell sensitivity of these drugs a role for p53 has been proposed at least for cisplatin and BBR 3464. Our results indicate that, also in the case of oxaliplatin, cytotoxicity is modulated by this pathway. Indeed, the effect of oxaliplatin could be reduced in tumor cells expressing mutant p53. The DNA mismatch repair system also appears to be critical in regulating cellular sensitivity to cisplatin because the loss of DNA mismatch repair results in low level of resistance to cisplatin, but not to oxaliplatin. Thus, platinum compounds are endowed with differential capability to activate pathways of p53-dependent or independent apoptosis, and differential recognition by specific cellular systems is likely to be the critical determinant of the cell fate (death/survival) after drug exposure. Further molecular studies are required to better define the precise contribution of such pathways to the cellular responses of the clinically relevant platinum complexes. A complete understanding of the molecular basis of sensitivity to platinum drugs is expected to provide useful insights for the optimization of tumor treatment.

The muscle nicotinic acetylcholine receptor (AChR) turns over at different rates depending on stage of synaptogenesis and innervation. Tyrosine phosphorylation modulates desensitization, interaction with cytoskeleton and lateral mobility in the membrane of AChR. To determine whether tyrosine phosphorylation also modulates the turnover of AChR, myotubes in vitro were exposed to the tyrosine phosphatase inhibitor pervanadate. Our data indicate that a transient increase of phosphotyrosine levels stabilized a fraction of AChRs. The effects were limited to the non-epsilon subunit-containing AChRs already present in the membrane. Tyrosine phosphorylation of the receptor occurred on the beta subunit, was transient and stable molecules were not selectively tyrosine phosphorylated. The data indicate that modulation of phosphotyrosine levels in muscle cells provides signals to control AChR metabolic stability.

Synchronous oscillations of intracellular calcium concentration ([Ca2+]i) and of membrane potential occurred in a limited population of glutamatergic hippocampal neurons grown in primary cultures. The oscillatory activity occurred in synaptically connected cells only when they were in the presence of astrocytes. Microcultures containing only one or a few neurons also displayed oscillatory activity, provided that glial cells participated in the network. The glutamate-transporter inhibitors L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC) and dihydrokainate, which produce an accumulation of glutamate in the synaptic microenvironment, impaired the oscillatory activity. Moreover, in neurons not spontaneously oscillating, though in the presence of astrocytes, oscillations were induced by exogenous L-glutamate, but not by the stereoisomer D-glutamate, which is not taken up by glutamate transporters. These data demonstrate that astrocytes are essential for neuronal oscillatory activity and provide evidence that removal of glutamate from the synaptic environment is one of the major mechanisms by which glial cells allow the repetitive excitation of the postsynaptic cell.

International audience; : Allogeneic stem cell (SC)-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some SC types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ectomesenchymal SCs, leptomeningeal SCs, and three different c-Kit-positive SC types, that is, amniotic fluid SCs, cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers ICAM-1, VCAM-1, HLA-ABC, and HLA-DR, modulatory activity toward purified T, B, and NK cells, lower immunogenicity of inflammatory-primed SCs as compared to resting SCs, and indoleamine-2,3-dioxygenase-activation as molecular inhibitory pathways, with some SC type-related peculiarities. Moreover, the SC types analyzed exert an anti-apoptotic effect toward not-activated immune effector cells (IECs). In addition, we found that the inhibitory behavior is not a constitutive property of SCs, but is acquired as a consequence of IEC activation, as previously described for MSCs. Thus, immune regulation is a general property of SCs and the characterization of this phenomenon may be useful for a proper therapeutic use of SCs.

Introduction: Before the nerve contacts the skeletal muscle, the nicotinic acetylcholine receptors (nAChRs) form aggregates known as prepatterned clusters. We investigated their role in the occurrence of Ca2+ spikes and twitching during myogenesis. Methods: Cultured mouse myotubes were used as cell models. Cells were subjected to a combination of immunostaining, Ca2+ imaging and electrophysiological analysis. Results: A single prepatterned nAChR cluster per myotube was generally detected. A correlation between formation of the prepatterned clusters and occurrence of Ca2+ spikes and twitching was observed. Increase in size of the prepatterned clusters raised the frequency of Ca2+ spikes and twitching. Blockade of the electrical activity triggered by the autocrine activation of prepatterned nAChR induced over-numbered nAChR clusters. Conclusions: Prepatterned nAChR aggregation is required for Ca2+ spikes and twitching of developing myotubes. Moreover, prepatterned nAChR-driven electrical activity preserves the distribution of nAChRs, mimicking the effect of synaptic activity before innervation. Muscle Nerve 46: 112–121, 2012

In vitro findings suggested a role for the p75 neurotrophin receptor in the maturation of GABAergic neurons residing in the basal forebrain (BF), a brain area known to have p75 expression only on cholinergic neurons. We document here the presence of GABAergic neurons which express p75 in the BF in vivo. Colocalization of p75 with the cholinergic marker choline-acetyltransferase (ChAT) and/or the GABAergic marker glutamic acid decarboxylase-67 (GAD67) was investigated in the BF at birth, at two weeks, and in adulthood. A subset of GAD67(+) neurons was p75(+) (p75(+)/GAD67(+)) but ChAT(-) in the substantia innominata and nucleus basalis magnocellularis at birth, whereas all p75(+)/GAD67(+) neurons were also ChAT(+) from two weeks onward. These phenotypic features suggest that a subpopulation of GABAergic neurons could be sensitive to neurotrophins during brain maturation. To unravel this issue, we then pursued a functional analysis by assessing p75 expression profile, and its modulation by nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) in primary BF cell cultures. NGF increased p75 expression exclusively in cholinergic neurons, whereas BDNF induced p75 expression only in a subset of GABAergic neurons (p75(+)/GAD67(+)/ChAT(-)) through a p75- and tyrosine-kinase-dependent mechanism. The latter findings point to a selective role of BDNF in the induction of p75 expression in BF GABAergic neurons. Altogether these results confirm the role of neurotrophins in the developing and mature circuitry of GABAergic neurons in the BF regions.

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury.

The effect of electrical stimulation on the stability of junctional ACh receptors (AChR) on soleus muscles of Wistar rats was compared to that of denervation and reinnervation. Denervation causes the degradation rate of the slowly degrading AChRs (Rs) at the neuromuscular junction to accelerate and be replaced by rapidly degrading AChRs (Rr), while reinnervation restabilizes the accelerated Rs. Electrical stimulation initiated at the time of denervation prevented the acceleration of the Rs. It could not, however, reverse the effect of denervation if initiated after the AChRs became destabilized, nor could it slow the degradation rate of the Rr. We conclude that electrical stimulation of denervated muscle downregulates the expression of the Rr and prevents the destabilization of Rs.

Introduction Expansion of affordances and acquisition of new possibilities for action depend on motor learning and development. Adolph considers affordances as continuous, probabilistic functions, representing an individual's likelihood of successful performance, across environmental increments. With this study we addressed the question of how the relation between educator and child in a difficult motor task, involving a process of expanding affordances, contributes to the acquisition of new possibilities for action [1] , [2] , [3] . Methods Sixty children 5 years old from 2 kindergartens in Treviso (Italy) were studied while performing structured and unstructured physical activity during one hour lessons at a common playground; frequency of lessons was 1/week and the total period of observation was for three months. Children of group A could rely on a teacher to get on a very difficult tool, a bar supported by two mobile springs; the teacher told them that they could jump down if they lost the balance or were scared of walking the bar. After the jump, they were encouraged to go up again at the same point of the bar. Children of group B received no help and were left alone to find out the best strategy to accomplish the task. Time and number of errors were measured and children were video recorded. Results At the beginning no children were able to climb on the bar. At the end of the 10 lessons, 20% only of the children of group B were able to climb on the bar by themselves, and none was able to walk. In group A, 80% of the children climbed on the bar by themselves, 17% requested help and three % could not climb. During free play, only children of group A used the spring bar. Conclusions The data indicate that teacher-mediated experiences promote child expansion of body scale and affordances. The data also suggest that successful experiences encourage autonomous child training thus further expanding perception of motor skill competence.

In this study, we investigated whether the potential positive effects of nicotine in Alzheimer's disease (AD) may involve neurotrophic factors, such as nerve growth factor (NGF), closely associated with basal forebrain (BF) cholinergic function and survival. To this aim, we studied the effects of prolonged nicotine treatment on neurotrophin receptors expression and on NGF protein levels in the rat BF cholinergic circuitry. Both in vivo and in vitro experiments were conducted. We found that s.c. nicotine infusion (1.2 mg free base/kg/d delivered by mini-pumps for 7 days) induced in vivo an increase in tyrosine kinase receptor A (TrkA)-but not TrkB, TrkC or low affinity neurotrophin receptor p75 (p75)-expression in BF cholinergic neurons targeting the cerebral cortex. Nicotine did not produce statistically significant long-lasting effects on NGF levels in the cerebral cortex, or in the BF. In vitro experiments performed on primary BF neuronal cultures, showed that 72 h exposure to nicotine increased both TrkA expression, and NGF release in culture medium. Neutralization experiments with an anti-NGF antibody showed that NGF presence was not necessary for nicotine-induced increase of TrkA levels in cultured cholinergic neurons, suggesting that nicotine may act through NGF-independent mechanisms. This study shows that nicotine, independently of its action on NGF levels, may contribute to the restoration of the trophic support to BF cholinergic neurons by increasing TrkA levels.

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve–muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and β-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

Noradrenergic transmission has been implicated in the affective component of relapse to tobacco smoking. Evidence in human and laboratory animals showed that smoking or nicotine administration may cause changes of the noradrenergic system resulting in hyperactivity in this system after cessation. It has been hypothesised that the anti-adrenergic β-blocker propranolol may decrease affective activation and arousal observed during drug withdrawal or cue-induced relapse. The aim of the present work was to test the effects of propranolol pre-treatment in a rat model of nicotine cue-induced relapse to nicotine seeking. We also tested the effects of propranolol on food cue-induced reinstatement of food seeking in rats trained on food self-administration. Propranolol transiently inhibited nicotine cue-induced reinstatement. The inhibitory effect of propranolol reached a peak after 30 min from the beginning of the reinstatement session and then it declined until it was completely absent at the end of the 3-h session. This inhibitory effect of propranolol was not observed when the drug was tested versus reinstatement with food cues. The present study suggests a weak effect of propranolol to counteract nicotine cue-induced reinstatement of nicotine seeking. Therefore, these findings do not support a potential use of propranolol for prevention of smoking relapse.

Release of Ca2+ from intracellular stores was studied in the parent PC12 cell line and in recently isolated clones sensitive or insensitive to caffeine. In the caffeine-sensitive cells the cytosolic free Ca2+ concentration ([Ca2+]i) responses by the xanthine drug and by stimulants of receptors coupled to inositol 1,4,5-trisphosphate (Ins-P3) generation (bradykinin, ATP) depend on separate pathways because 1) caffeine does not stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate and 2) Ca(2+)-induced Ca2+ release, the process activated by caffeine, plays no major role in the Ins-P3-induced Ca2+ mobilization. Although distinct, these two mechanisms converge onto the same Ca2+ store. In fact 1) the [Ca2+]i responses by receptor agonists and caffeine were not additive; 2) either type of agent reduced (up to complete inhibition) the response to a subsequent administration of the same or the other agent; 3) all these responses were prevented by selective Ca2+ ATPase blockers; 4) ryanodine, which affects the intracellular Ca2+ channel sensitive to caffeine, also induced depletion of the receptor-sensitive Ca2+ pool; 5) in the 10 PC12 clones tested, sensitivity to caffeine paralleled ryanodine sensitivity. Therefore, PC12 cells, similar to some smooth muscle fibers but at variance with neurons and other secretory cells, express a single, rapidly exchanging Ca2+ store in which two distinct intracellular Ca2+ channels, i.e. the receptors for caffeine-ryanodine and Ins-P3, appear to be colocalized.

At the normal mammalian neuromuscular junction the half-life of the acetylcholine receptor (AChR) ranges from 6 to 13 days (estimates from seven different laboratories). Indirect evidence suggests that the internalized receptor is degraded by a lysosomal mechanism. We have now traced the fate of the AChR labelled in vivo with peroxidase-alpha-bungarotoxin. Segments of junctional folds bearing AChRs are internalized by endocytosis. The endocytosed vesicles are engulfed by tubules and larger vesicles which, by electron cytochemical criteria, represent secondary lysosomes. Pathological mechanisms increased AChR loss from the end-plate. These include destruction of junctional folds, formation of immature junctions with a few or no junctional folds, accelerated internalization of AChR, impaired membrane insertion of new AChR and, possibly decreased AChR synthesis. The common mechanism for destruction of the junctional folds is an altered subsynaptic ionic milieu, and especially focal calcium excess. This can be induced by antibody and complement, too frequent or prolonged openings of the acetylcholine (ACh)-induced ion channel, and other membrane defects. In acquired autoimmune myasthenia gravis there is (a) antibody-dependent complement-mediated lysis of the junctional folds, (b) accelerated internalization of AChR cross-linked by antibody and (c) decreased insertion of AChR into the postsynaptic membrane. The last mechanism is attributed to lack of membrane patches available for tight packing and secure anchoring of the receptor. In acute, but not in chronic, experimental autoimmune myasthenia gravis, and infrequently in human myasthenia gravis, macrophages destroy junctional folds opsonized by antibody and C3. In a recently recognized congenital syndrome attributed to a prolonged open time of the ACh-induced ion channel, and to a lesser extent in congenital end-plate acetylcholinesterase deficiency, AChR is lost with degradation of junctional folds. In other, less well-defined, congenital syndromes there is deficiency or abnormal function of AChR. This could arise from decreased synthesis or membrane insertion or accelerated degradation of AChR, or from a structurally abnormal AChR with reduced affinity for ACh or with a diminished conductance or open time of its ion channel.

Recent investigations suggest that the effects of neural agrin might not be limited to neuromuscular junction formation and maintenance and that other aspects of muscle development might be promoted by agrin. Here we tested the hypothesis that agrin induces a change in the excitability properties in primary cultures of non-innervated human myotubes. Electrical membrane properties of human myotubes were recorded using the whole-cell patch-clamp technique. Cell incubation with recombinant chick neural agrin (1 nM) led to a more negative membrane resting potential. Addition of strophanthidin, a blocker of the Na(+)/K(+) ATPase, depolarized agrin-treated myotubes stronger than control, indicating, in the presence of agrin, a higher contribution of the Na(+)/K(+) ATPase in establishing the resting membrane potential. Indeed, larger amounts of both the alpha1 and the alpha2 isoforms of the Na(+)/K(+) ATPase protein were expressed in agrin-treated cells. A slight but significant down-regulation of functional apamin-sensitive K(+) channels was observed after agrin treatment. These results indicate that neural agrin might act as a trophic factor promoting the maturation of membrane electrical properties during differentiation, confirming the role of agrin as a general promoter of muscle development.

The type 3 small conductance calcium-activated potassium channel (SK3) is expressed in embryonic and adult denervated skeletal muscles where it contributes to hyperexcitability. This study aimed at determining the role of muscle activity in regulating SK3 channels. Soleus muscles of adult rats were denervated by cutting the sciatic nerve. In reinnervation studies, the soleus nerve was crushed: in one group, muscles were reinnervated with electrically silent axons, by chronic sciatic nerve perfusion with tetrodotoxin. Several groups of denervated muscles were subjected to chronic direct electrical stimulation, using either fast (100 Hz) or slower patterns (20 or 30 Hz). The SK3 mRNA and protein levels in soleus muscle were determined by reverse transcriptional-PCR, Western blot and immunofluorescence. Both denervated and reinnervated–paralysed soleus muscles displayed similar up-regulation of SK3 mRNA and protein. Reinnervation with electrically active axons instead inhibited SK3 up-regulation. Chronic muscle direct stimulation in vivo, irrespective of the pattern used, reversed the denervation-induced up-regulation of SK3 expression or prevented it when initiated at the time of denervation. Chronic electrical stimulation of denervated muscles also completely prevented the development of the after-hyperpolarization (AHP) following the action potential, normally induced in the muscle fibres by denervation. We conclude that action potential activity evoked by motor neurones in muscle fibres is both necessary and sufficient to account for the physiological down-regulation of SK3 channels in the non-junctional membrane of skeletal muscle.

This work investigated in rats whether passive immunization against the endogenous GHRF in the early postnatal period led to permanent alterations of somatotropic function, similar to those observed in several human growth disorders, e.g. constitutional growth delay (CGD). On postnatal days 1, 2, 4, 6, 8, and 10, rats were given an anti-GHRF-serum (GHRH-Ab, 100 microliters/rat, sc) and were tested 1, 30, and 60 days after this treatment for basal and GHRH-stimulated GH secretion both in vivo and in vitro. GHRH-Ab reduced both basal and GHRF-stimulated GH secretion at all intervals and induced marked and chronic impairment of growth rate. The following differences were observed in the GHRH-Ab treated rats compared to normal rabbit serum-treated controls: 1) GH biosynthesis (incorporation of L-[3H]leucine into the electrophoretic band of GH): reduction of about 70%, 1 day but not 30 days after treatment; 2) Pituitary weight: significant reduction in absolute weight (30-40%) at all posttreatment intervals, and relative weight, 1 and 30 days after treatment. 3) Pituitary GH concentration: significant reduction in GH content (about 40%) but not concentration, at all posttreatment intervals; 4) Percentage of somatotrophs (immunocytochemistry): about 40% reduction 1 day, but not 30 and 60 days after treatment; 5) Hypothalamic somatostatin messenger RNA (mRNA) levels in situ hybridization): selective reduction (40%) in the periventricular nucleus 1 day but not 30 days after treatment; 6) Hypothalamic somatostatin cell number (immunocytochemistry): no significant changes in any hypothalamic area at any interval; 7) Pituitary somatostatin binding (in situ autoradiography): significant reduction, 1 day and 30 days after treatment; 8) Somatostatin inhibition of GH release "in vitro": somatostatin effect on GH release was reduced 30 days after treatment. These and previous data indicate that: 1) Transient deprivation of GHRF in the immediate postnatal period of the rat leads to permanent impairment of growth rate and somatotropic function; 2) GHRF deficiency itself or through reduction of GH secretion impairs somatostatin functions temporarily in the hypothalamus and permanently in the pituitary; 3) This rat model may mimic some forms of growth disorders in humans and holds promise as useful tools for investigating the underlying pathophysiological mechanisms.

The scope of this study was to test whether increased levels of the extracellular matrix molecule (ECM) agrin might enhance nicotine effects on those molecular mechanisms that initiate neuroadaptative processes in the hippocampus, a key brain area for learning and memory. We studied the effects of repetitive applications of neuronal agrin to primary hippocampal cell culture on nicotine-induced phosphorylated cyclic AMP response element-binding protein (pCREB) expression, a marker of neuroadaptation, by using immunofluorescence-based assessment of pCREB-positive neurons. We also tested agrin effects on nicotine-induced expression of a marker of metabolic activation, the immediate early gene c-fos. Agrin was shown to significantly enhance nicotine-induced pCREB, but not c-fos, expression. By using Western blotting analysis, cumulative agrin has been shown to increase nicotine-induced pCREB phosphorylation. These analyses, however, showed that inhibition of the CaMKII pathway blocked general pCREB phosphorylation, whereas inhibition of the MAPK pathway potentiated the synergistic effect of cumulative agrin and nicotine. These findings suggest that increasing the concentration of an ECM molecule, i.e. agrin, may enhance nicotine effects on pCREB and that both MAPK and CaMKII signalling may play a regulatory role.

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca2+ imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca2+ channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca2+ channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.

This preliminary study describes magnetic resonance imaging (MRI) data on the effects of sub-chronic nicotine administration in rats. Nicotine 0.4 mg/kg s.c. free base was given once a day for 4 days to Wistar adult male rats. On day 5, anaesthetized subjects were observed using an MRI tomography system. Regional cerebral blood volume (rCBV) and transversal relaxation time (T2) MRI parameters were measured. Nicotine treatment increased T2 values, with a significant effect in the cingulate cortex. A trend to increase was also observed in the prefrontal cortex and nucleus accumbens. Similarly, the effect of nicotine on rCBV was a significant increase in values compared to saline treatment. Post hoc analysis showed a significant effect of nicotine in the prefrontal cortex, cingulate cortex, mediodorsal thalamus and lateral posterior thalamus. This study showed for the first time that sub-chronic nicotine administration can induce changes in MRI pattern which are (i) similar to human MRI studies, and (ii) common to those described for markers of neuronal metabolic activation in corticolimbic brain regions known to be involved in nicotine dependence.

Intracellular trafficking is an important event in the control of type and number of ion channels expressed on the cell surface. In this study, we have identified molecular domains involved in assembly and trafficking of the human small conductance Ca2+-activated K+ channel SK3. Deletion of the N-terminus, the C-terminus, or the calmodulin-binding domain (CaMBD) led to retention of SK3 channels in the endoplasmic reticulum. Presence of the CaMBD allowed trafficking to the Golgi complex, and sequences downstream were required for efficient transport to the plasma membrane, suggesting several steps in the control of SK3 forward trafficking. Co-immunoprecipitation studies demonstrated that SK3 subunits lacking the N-terminus, the CaMBD, or the distal C-terminus, but not the entire C-terminus, were able to oligomerize with wild-type SK3 subunits. Thus, these two C-terminal regions of SK3 seem to contribute to assembly and trafficking of channels whereas the N-terminus is necessary for trafficking but not sufficient for oligomerization.

At mammalian neuromuscular junctions (NMJs), innervation induces and maintains the metabolic stability of acetylcholine receptors (AChRs). To explore whether neural agrin may cause similar receptor stabilization, we injected neural agrin cDNA of increasing transfection efficiencies into denervated adult rat soleus (SOL) muscles. As the efficiency increased, the amount of recombinant neural agrin expressed in the muscles also increased. This agrin aggregated AChRs on muscle fibers, whose half-life increased in a dose-dependent way from 1 to 10 days. Electrical muscle stimulation enhanced the stability of AChRs with short half-lives. Therefore, neural agrin can stabilize aggregated AChRs in a concentration- and activity-dependent way. However, there was no effect of stimulation on AChRs with a long half-life (10 days). Thus, at sufficiently high concentrations, neural agrin alone can stabilize AChRs to levels characteristic of innervated NMJs.

Alternative splicing in the mGluR5 gene generates two different receptor isoforms, of which expression is developmentally regulated. However, little is known about the functional significance of mGluR5 splice variants. We have examined the functional coupling, subcellular targeting, and effect on neuronal differentiation of epitope-tagged mGluR5 isoforms by expression in neuroblastoma NG108-15 cells. We found that both mGluR5 splice variants give rise to comparable [Ca2+]i transients and have similar pharmacological profile. Tagged receptors were shown by immunofluorescence to be inserted in the plasma membrane. In undifferentiated cells the subcellular localization of the two mGluR5 isoforms was partially segregated, whereas in differentiated cells the labeling largely redistributed to the newly formed neurites. Interestingly, we demonstrate that mGluR5 splice variants dramatically influence the formation and maturation of neurites; mGluR5a hinders the acquisition of mature neuronal traits and mGluR5b fosters the elaboration and extension of neurites. These effects are partly inhibited by MPEP.