Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Vesicular acetylcholine transporter knock down-mice are more susceptible to inflammation, c-Fos expression and sickness behavior induced by lipopolysaccharide.

In addition to the well-known functions as a neurotransmitter, acetylcholine (ACh) can modulate of the immune system. Nonetheless, how endogenous ACh release inflammatory responses is still not clear. To address this question, we took advantage of an animal model with a decreased ACh release due a reduction (knockdown) in vesicular acetylcholine transporter (VAChT) expression (VAChT-KD(HOM)). These animals were challenged with lipopolysaccharide (LPS). Afterwards, we evaluated sickness behavior and quantified systemic and cerebral inflammation as well as neuronal activation in the dorsal vagal complex (DVC). VAChT-KD(HOM) mice that were injected with LPS (10mg/kg) showed increased mortality rate as compared to control mice. In line with this result, a low dose of LPS (0.1mg/kg) increased the levels of pro-inflammatory (TNF-α, IL-1ß, and IL-6) and anti-inflammatory (IL-10) cytokines in the spleen and brain of VAChT-KD(HOM) mice in comparison with controls. Similarly, serum levels of TNF-α and IL-6 were increased in VAChT-KD(HOM) mice. This excessive cytokine production was completely prevented by administration of a nicotinic receptor agonist (0.4mg/kg) prior to the LPS injection. Three hours after the LPS injection, c-Fos expression increased in the DVC region of VAChT-KD(HOM) mice compared to controls. In addition, VAChT-KD(HOM) mice showed behavioral changes such as lowered locomotor and exploratory activity and reduced social interaction after the LPS challenge, when compared to control mice. Taken together, our results show that the decreased ability to release ACh exacerbates systemic and cerebral inflammation and promotes neural activation and behavioral changes induced by LPS. In conclusion, our findings support the notion that activity of cholinergic pathways, which can be modulated by VAChT expression, controls inflammatory and neural responses to LPS challenge.

Reduced expression of VAChT increases renal fibrosis.

Chronic kidney disease (CKD) is associated with several other long-lasting conditions such as diabetes and cardiovascular diseases and it is a significant contributor to mortality worldwide. Obstructive kidney disease is one of the leading causes of CKD in children and may result from a wide variety of pathologic processes. Recent studies have shown that α7 nicotinic acetylcholine receptor (α7 nAChR) activation in the cholinergic anti-inflammatory pathway reduces production of inflammatory mediators and consequently prevents tissue injury and death. Here, we examined the role of endogenous release of acetylcholine on the development of fibrosis in renal tissue using a model of unilateral ureter obstruction (UUO)-induced CKD, in which obstruction promotes inflammation-mediated kidney damages. To interfere with acetylcholine secretion, we used mice in which the vesicular acetylcholine transporter is genetically reduced (VAChT KD(hom) mice). We observed a higher renal damage in VAChT mutant mice when compared to wild type controls, exemplified by higher proteinuria and increased amount of type 1 collagen in the kidney tissue, indicating accentuated fibrogenesis. These results were accompanied by enhanced localized kidney inflammation, with increased TH1/TH17 profile response. Administration of PNU-282987, a selective agonist of α7 nAChR, significantly attenuated kidney injury after UUO in VAChT KD(hom) mice, indicating that the lack of acetylcholine release decrease the action of the cholinergic anti-inflammatory pathway, promoting an up-regulation of pro-inflammatory and pro-fibrotic pathways. These results suggest that physiological activation of the cholinergic anti-inflammatory pathway regulates inflammatory responses in the kidney suggesting a new therapeutic approach for kidney disease.

Autonomic nervous system modulation affects the inflammatory immune response in mice with acute Chagas disease.

The aim of the present study was to evaluate the effects of changes to the autonomic nervous system in mice during the acute phase of Chagas disease, which is an infection caused by the parasite Trypanosoma cruzi. The following types of mice were inoculated with T. cruzi (CHG): wild-type (WT) and vesicular acetylcholine transporter knockdown (KDVAChT) C57BL/6j mice; wild-type non-treated (NT) FVB mice; FVB mice treated with pyridostigmine bromide (PYR) or salbutamol (SALB); and ß(2)-adrenergic receptor knockout (KOß2) FVB mice. During infection and at 18-21 days after infection (acute phase), the survival curves, parasitaemia, electrocardiograms, heart rate variability, autonomic tonus and histopathology of the animals were evaluated. Negative control groups were matched for age, genetic background and treatment. The KDVAChT-CHG mice exhibited a significant shift in the electrocardiographic, autonomic and histopathological profiles towards a greater inflammatory immune response that was associated with a reduction in blood and tissue parasitism. In contrast, the CHG-PYR mice manifested reduced myocardial inflammation and lower blood and tissue parasitism. Similar results were observed in CHG-SALB animals. Unexpectedly, the KOß2-CHG mice exhibited less myocardial inflammation and higher blood and tissue parasitism, which were associated with reduced mortality. These findings could have been due to the increase in vagal tone observed in the KOß2 mice, which rendered them more similar to the CHG-PYR animals. In conclusion, our results indicate a marked immunomodulatory role for the parasympathetic and sympathetic autonomic nervous systems, which inhibit both the inflammatory immune response and parasite clearance during the acute phase of experimental Chagas heart disease in mice.

Aerobic exercise training engages cholinergic signaling to improve emphysema induced by cigarette smoke exposure in mice.

Lung inflammation is modulated by cholinergic signaling and exercise training protects mice against pulmonary emphysema development; however, whether exercise training engages cholinergic signaling is unknown. AIMS: As cholinergic signaling is directly linked to the vesicular acetylcholine transporter (VAChT) levels, we evaluated whether the effects of aerobic exercise training depend on the VAChT levels in mice with pulmonary emphysema. MAIN METHODS: Wild-type (WT) and mutant (KDHOM) mice (65-70% of reduction in VAChT levels) were exposed to cigarette smoke (30 min, 2×/day, 5×/week, 12 weeks) and submitted or not to aerobic exercise training on a treadmill (60 min/day, 5×/week, 12 weeks). Lung function and inflammation were evaluated. KEY FINDINGS: Cigarette smoke reduced body mass in mice (p < 0.001) and increased alveolar diameter (p < 0.001), inflammation (p < 0.001) and collagen deposition (p < 0.01) in lung tissue. Both trained groups improved their performance in the final physical test compared to the initial test (p < 0.001). In WT mice, exercise training protected against emphysema development (p < 0.05), reduced mononuclear cells infiltrate (p < 0.001) and increased MAC-2 positive cells in lung parenchyma (p < 0.05); however, these effects were not observed in KDHOM mice. The exercise training reduced iNOS-positive cells (p < 0.001) and collagen fibers deposition (p < 0.05) in lung parenchyma of WT and KDHOM mice, although KDHOM mice showed higher levels of iNOS-positive cells. SIGNIFICANCE: Our data suggest that the protective effects of aerobic exercise training on pulmonary emphysema are, at least in part, dependent on the integrity of the lung cholinergic signaling.

Histamine H3 Receptors Decrease Dopamine Release in the Ventral Striatum by Reducing the Activity of Striatal Cholinergic Interneurons.

Histamine H3 receptors are widely distributed Gi-coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H3 agonist α-methylhistamine significantly reduced electrically- evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-ß-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H3-modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H3 receptors by α-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by α-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by α-methylhistamine. Together, these results indicate that histamine H3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons.

A rat homologue of CED-6 is expressed in neurons and interacts with clathrin.

We isolated from a brain library a cDNA encoding an isoform of rat CED-6 that has not been previously described. This transcript results from alternative splicing of the ced-6 gene present on chromosome 9. We expressed this isoform as his-tagged protein in E. coli and used the purified protein to raise antibodies to investigate the expression of CED-6 in rat brain. Immunoblot analysis showed the presence of CED-6 as a doublet of approximately 34 and 33 kDa in cortex, hippocampus and cerebellum, indicating that the protein was present in different regions of the brain. Subcellular fractionation experiments showed that CED-6 immunoreactivity did not concentrate in GFAP-containing glial vesicles, whereas it showed a distribution similar to the synaptotagmin in synaptosomes-enriched fractions, suggesting that CED-6 is present in neurons. CED-6 immunoreactivity was also investigated using immunohistochemistry analysis and it was found in several brain regions, being particularly strong in the cell body of some groups of neurons such as Purkinje cell layer of cerebellum, and pyramidal cells of the hippocampal formation and also in epithelial cells from the choroid plexus. Importantly, CED-6 immunoreactivity colocalized with a neuronal marker but not with a glial marker. Considering that several PTB-containing proteins bind clathrin, we investigated whether rat CED-6 would also have this property. Yeast two-hybrid and GST pull-down analysis indicated that ratCED-6 interacts with clathrin and in cultured cells we detected colocalization between CED-6 and clathrin-coated vesicles. The present findings suggest that CED-6 may have a role in endocytic trafficking or signaling in neurons.

Endogenous Acetylcholine Controls the Severity of Polymicrobial Sepsisassociated Inflammatory Response in Mice.

Acetylcholine (ACh) is the main mediator associated with the anti-inflammatory cholinergic pathway. ACh plays an inhibitory role in several inflammatory conditions. Sepsis is a severe clinical syndrome characterized by bacterial dissemination and overproduction of inflammatory mediators. The aim of the current study was to investigate the participation of endogenous ACh in the modulation of inflammatory response induced by a model of polymicrobial sepsis. Wild type (WT) and vesicular acetylcholine transporter knockdown (VAChT(KD)) mice were exposed to cecal ligation and perforation- induced sepsis. Levels of Tumor Necrosis Factor Alpha (TNF-α) and bacterial growth in peritoneal cavity and serum, and neutrophil recruitment into peritoneal cavity were assessed. The concentration of TNF-α in both compartments was higher in VAChT(KD) in comparison with WT mice. VAChT(KD) mice presented elevated burden of bacteria in peritoneum and blood, and impairment of neutrophil migration to peritoneal cavity. This phenotype was reversed by treatment with nicotine salt. These findings suggest that endogenous ACh plays a major role in the control of sepsis-associated inflammatory response.

Reduced expression of mir15a in the blood of patients with oral squamous cell carcinoma is associated with tumor staging.

MicroRNAs (miRNAs) mir15a and let7a are important regulators of bcl-2, ras and c-myc proteins. Considering that these miRNAs are commonly altered in many human cancers and that these proteins are reported to be altered in oral squamous cell carcinoma (OSCC), we investigated them in a set of OSCC cases. The miRNAs as well as the proteins were evaluated in the tumor and blood of 20 patients by real-time quantitative PCR and immunohistochemistry, respectively. The expression of mir15a and bcl-2 proteins in the tumors was not associated with each other or with tumor staging. On the other hand, we found reduced expression of this miRNA in the blood of patients with an advanced stage of OSCC and with lymph node metastasis. The expression of let7a in the tumor and blood was not associated with tumor size, lymph node metastasis, tumor staging and immunoexpression of ras and c-myc proteins. In conclusion, the present study shows that reduced expression of mir15a is associated with OSCC staging.