1,721,061 research outputs found
In vivo acetylcholine receptor expression induced by calcitonin gene-related peptide in rat soleus muscle
No full textWe applied calcitonin gene-related peptide (CGRP) by continuous perfusion of the extrajunctional surface of the adult rat soleus muscle in vivo. We obtained this through a fine polyethylene catheter connected to an Alzet pump implanted in the animal. The perfusion induced a local acetylcholine receptor accumulation in the membrane of the muscle fibres starting with a delay of one to two days, provided a chronic conduction block of soleus innervation was concomitantly present. The effect was prominent, being higher than that following denervation. The lack of acetylcholine receptor accumulation observed in sham perfused animals and the co-administration of CGRP and its competitive antagonist peptide, hCGRP(8-37), eliminates the possibility that the response to CGRP application represents an inflammatory reaction to foreign bodies instead of a specific effect of the peptide.We suggest that CGRP may act on the extrajunctional membrane of muscle fibres to help induce acetylcholine receptor accumulation after appropriate receptors for the peptide are re-expressed due to muscle paralysis. Whilst this is compatible with a role of CGRP in synaptogenesis, a recent study showed that alpha-CGRP(-/-) mutant mice have normal neuromuscular junction development. However, given the redundancy of factors involved in acetylcholine receptor accumulation, further experiments on multiple knock-outs need to be performed before a final conclusion is reached about the physiological significance of CGRP
Analysis of antibody microarrays high throughput data: from data processing to network inference. The case of B-cell chronic lymphocytic leukemia.
No full textA particular category of protein arrays, namely antibody microarrays, is becoming increasingly important in proteomic research. Consistent developments in the least few years led to the possibility to detect expression and phosphorylation level of hundreds of proteins at once in biological samples with unprecedented sensitivity, still unmatched by other methods, such as mass-spec. Being a relatively new technique, and considering the wealth of information potentially generated, the antibody microarrays approach requires ad-hoc strategies in terms of signal quantification and filtering, data normalization, statistical analysis and functional interpretation. In this study, several aspects are discussed, with particular focus on the antibody microarrays technology developed by the company Kinexus, CA USA.A meaningful example of the application of the discussed criteria is presented, in the context of a large study of human leukemia samples we performed, including 34 B-cell chronic lymphocytic leukemia and 10 healthy human subjects before and after stimulation with the chemokine CXCL12, analyzed by means of 44 arrays monitoring over 800 antibodies, also including about 300 antibodies detecting phosphosites. Comparison strategies and network analysis approaches are described. We used the network analysis platform Cytoscape (http://www.cytoscape.org/) along with plugins developed by our group (PesCa, CentiScaPe, www.cbcm.it) to highlight key pathways suggested by array data. Particular emphasis is given to the impact of microarray data on proteins network reconstruction and understanding of fundamental biological processes
Physical activity and anodal-transcranial direct current stimulation: a synergistic approach to boost motor cortex plasticity
Full text linkThe application of anodal-transcranial direct current stimulation (A-tDCS) over the primary motor cortex (M1) increases its structural and functional plasticity, as also physical exercise. Combining both interventions has a boosting effect, thus revealing a crucial role of the brain state during stimulation. Although brain slice and anesthetized animal studies support this, further investigation in awake animals is necessary. In the present study, we analyzed the effects of coupling A-tDCS with low-intensity physical activity on the mouse M1 structural and functional plasticity. C57BL/6 mice were monolaterally treated with M1 A-tDCS while walking on a rotarod or at rest. To assess the impact of our interventions, we analyzed both motor cortices for changes in neuronal activation, dendritic spine density, and functional synchronisation as measured by local field potential coherence. The combination of physical activity and M1 stimulation revealed a synergistic interhemispheric effect on cortical activation in both layers II/III and V, not present when using a single type of intervention. These data were accompanied by increased M1-M1 synchrony in the low-theta frequency, a hallmark of motor network activity in mice. Dendritic spine density revealed an effect of the combo, which was significantly higher only in layer II/III, accompanied by increased post-synaptic density protein 95 expression in the same area. Based on our findings, we propose that the efficacy of tDCS hinges on brain state rather than being merely a direct causal factor. The observed outcomes contribute to a deeper comprehension of the mechanisms governing structural and functional reorganisation within the motor cortex under physiological conditions, with potential implications for research on learning, memory, and neurological disorders such as stroke
Paralysis of innervated and reinnervated muscles equally affects contractile properties as does permanent denervation
No full textThe effects of long lasting (4--5 weeks) nerve conduction block and denervation were compared by investigating contractile, morphological and histochemical properties of slow (soleus) and fast (EDL) rat skeletal muscles. The block was based on improved perfusion techniques of the sciatic nerve with a tetrodotoxin (TTX) solution delivered at doses adequate to obtain maximal effects in the muscles. The TTX-inactivated axons retained normal histological and physiological properties such as the ability to evoke full contractile responses, to regenerate, and to completely reinnervate muscle. In spite of their intact innervation or of their full reinnervation, the TTX-paralysed muscles underwent weight loss, fibre atrophy and reduction in force output quantitatively indistinguishable from those following denervation. The same was true for all other contractile parameters tested, that is, twitch speed, twitch to tetanus ratio, post-tetanic potentiation, endurance, and fibre type composition. The results indicate the fundamental role of activity as a regulatory signal for muscle contractile properties, while they do not support the notion of a participation of chemical, activity-independent factors in this regulatio
On the mechanism of action of muscle fibre activity in synapse competition and elimination at the mammalian neuromuscular junction.
No full textActivity-dependent competition plays a crucial role in the refinement of synaptic connections in the peripheral and central nervous system. The reduction in number of axons innervating each neuromuscular junction during development, i.e. synapse elimination, appears to be one such competitive activity-driven event. Recently, we showed that asynchronous firing of competing presynaptic terminals is a key player in synapse elimination. Although some previous studies suggested that activity of the postsynaptic cell may be an intermediary in the disposal of redundant presynaptic inputs, the mechanism involved remains unknown. In the present study, in order to assess the role of evoked muscle activity in this process, we inhibited the generation of postsynaptic action potentials in muscle fibers in vivo, through the overexpression of inwardly rectifying Kir2.1 and Kir2.2 channels, via electroporation of the soleus muscle in the mouse hindlimb. Electrophysiological and morphological data show that overexpression of potassium channels in the endplate region of neonatal muscle fibres induces membrane hyperpolarization and an increase in conductance, inhibition of the action potential mechanism and prolonged persistence of polyneuronal innervation. These changes are not seen in muscle fibres with overexpression of a non-conducting Kir2.1 mutant. Our results are compatible with the interpretation that the block of action potential generation, even in single endplates, can inhibit synapse elimination through local signalling
Role of glucocorticoid receptors on dendritic spine plasticity in an Alzheimer’s disease animal model
No full textAIMS: Glucocorticoids hormones regulate stress response and are implicated in numerous diseases. The aim of our research is to assess the role of glucocorticoids in the pathogenesis of Alzheimer’s Disease (AD) and, in particular, to evaluate how the administration of agonists and antagonist of glucocorticoid receptors (GR) alter synaptic plasticity, causing a remodeling of dendritic spines in an AD mouse model (3xTg). METHOD: We administered either dexamethasone, an agonist of GR, or mifepristone, an antagonist of GR, to 6 month old 3xTg male mice. After perfusion with paraformaldehyde, brains were processed for Golgi Cox staining to highlight dendritic spines in a subset of neurons. We acquired images, reconstructed dendrites and, finally, calculated the dendritic spine density using the Imaris software (Bitplane). RESULTS: We found that dexamethasone causes a 23% reduction of spine density in CA1 region of hippocampus, whereas mifepristone resulted in a 14% significant increase, together with a modification of spine morphology. CONCLUSION: Dexamethasone-dependent reduction of spines is bigger than the one found in literature for treated wild type mice, suggesting a synergy between the molecular pathways leading to AD and the molecular action of glucocorticoids. Considering mifepristone treatment results, the blocking of GR could represent a possible therapeutic approach to slow down the progression of AD. In further studies, we will perform the experiments with dexamethasone and mifepristone to 10-11 months old 3xTg male mice to evaluate their effects on brains also in the late phase of pathology and we will investigate the molecular mechanism underlying the dendritic spine density changes
The timing of impulse activity shapes the process of synaptic competition at the neuromuscular junction.
No full textThe development of neuromuscular junctions exhibits profound remodeling that brings from an immature state characterized by multiple motoneuronal inputs per muscle fiber, to a mature mononeuronal innervation. This striking elimination process occurs both perinatally and during adult reinnervation, and is also widely present in the developing CNS. The accelerating influence of the amount of impulse activity on this process, has been shown by various studies, but a more subtle role of the time correlation of action potential firing in the competing inputs, has also been suggested. Here we explore the latter influence using a rat adult model of neuromuscular junction formation, that is reinnervation following a motor nerve crush. This shares all important features with perinatal development, especially the strict juxtaposition of the competing inputs, since. In fact the regenerating axons converge on a single cluster of postsynaptic receptors, that is the original synaptic site endplate of each muscle fiber. A similar focus on the spatial aspect of competition between nerve endings was missing in our previous experiments employing a similar paradigm. We impose a chronic synchronous firing to the competing terminals, by in vivo electrical stimulation of their axons distal to a sciatic nerve conduction block. Control preparations, with similar post-crush reinnervation, are left with their natural impulse activity unperturbed. We find that the experimental muscles display a prolonged duration of polyneuronal innervation with respect to controls, indicating that hebbian mechanisms participate in the synapse elimination process. Another aspect dealt with in our study is the genuine nature of the polyneuronal innervation occurring during adult muscle reinnervation, because it is supported by both confocal microscopy and by appropriate electrophysiological tests that exclude electrical coupling of myofibers by gap junctions
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