Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

International audienceMicroglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. METHODS: The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. FINDINGS: We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2-11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75-1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58-1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91-1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70-1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11-0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50-0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38-0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45-0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. INTERPRETATION: Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. FUNDING: Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health

Function of the voltage-gated Ca2+ channel 2-1 subunit in insulin release and diabetes

Type 2 diabetes occurs when an insufficient amount of insulin is released in the bloodstream, thus failing to maintain normal glycaemia. Pancreatic beta-cells are the main cell type residing in the pancreatic islets and are responsible for the release of insulin. beta-cells are excitable and express several voltage-gated ion channels and among them, voltage-gated Ca2+ channels are critical for the release of insulin. In fact, once a pancreatic beta-cell is depolarized, high voltage-gated Ca2+ channels activate, leading to Ca2+ influx and raise in cytosolic Ca2+ concentrations. The subsequent Ca2+ increase triggers the exocytic machinery and thus the release of insulin. Voltage-gated Ca2+ channels are composed by the pore-forming alpha1 subunit and by auxiliary subunits beta and alpha2delta. alpha2delta subunits regulate the membrane targeting of the alpha1 subunit and its current kinetics and they have shown their importance in a variety of neuronal and non-neuronal cell types. However, the importance of alpha2delta subunits for the release of insulin has never been previously reported. In this thesis, we investigated the role of the alpha2delta-1 subunit in pancreatic beta-cells. We have found that alpha2delta-1 is the main alpha2delta isoform expressed in pancreatic islets. Moreover, mice lacking the alpha2delta-1 subunits develop diabetes in a gender-dependent fashion. alpha2delta-1-/- male mice show a severe glucose intolerance, while alpha2delta-1-/- female mice are pre-diabetic, with a milder glucose intolerance. Pancreatic beta-cells from both alpha2delta-1-/- male and female mice have reduced Ca2+ influx. Nonetheless, only pancreatic islets from male alpha2delta-1-/- mice show reduced and delayed Ca2+ transients, while islets from female alpha2delta-1-/- mice have unaffected Ca2+ increase, however still delayed. Moreover, islets from alpha2delta-1+/+ female mice have reduced Ca2+ increase, compared to islets from alpha2delta-1+/+ male mice. We have shown that the differences in the Ca2+ transients between islets from male and female mice are related to a lower percentage of responding cells in islets from female mice. Despite the differences in the Ca2+ increase, pancreatic islets from both alpha2delta-1-/- male and female mice have reduced insulin release and, indeed, beta-cells from alpha2delta-1-/- male mice show reduced exocytosis. In addition, islets from female mice have increase insulin content, saving the alpha2delta-1-/- female mice from developing a severe diabetic condition. Together, these findings demonstrate the importance of the Ca2+ channel alpha2delta-1 subunit for the release of insulin and that mutations in alpha2delta-1 have the potential to be disease-causing in diabetic patients.Type 2 diabetes occurs when an insufficient amount of insulin is released in the bloodstream, thus failing to maintain normal glycaemia. Pancreatic beta-cells are the main cell type residing in the pancreatic islets and are responsible for the release of insulin. beta-cells are excitable and express several voltage-gated ion channels and among them, voltage-gated Ca2+ channels are critical for the release of insulin. In fact, once a pancreatic beta-cell is depolarized, high voltage-gated Ca2+ channels activate, leading to Ca2+ influx and raise in cytosolic Ca2+ concentrations. The subsequent Ca2+ increase triggers the exocytic machinery and thus the release of insulin. Voltage-gated Ca2+ channels are composed by the pore-forming alpha1 subunit and by auxiliary subunits beta and alpha2delta. alpha2delta subunits regulate the membrane targeting of the alpha1 subunit and its current kinetics and they have shown their importance in a variety of neuronal and non-neuronal cell types. However, the importance of alpha2delta subunits for the release of insulin has never been previously reported. In this thesis, we investigated the role of the alpha2delta-1 subunit in pancreatic beta-cells. We have found that alpha2delta-1 is the main alpha2delta isoform expressed in pancreatic islets. Moreover, mice lacking the alpha2delta-1 subunits develop diabetes in a gender-dependent fashion. alpha2delta-1-/- male mice show a severe glucose intolerance, while alpha2delta-1-/- female mice are pre-diabetic, with a milder glucose intolerance. Pancreatic beta-cells from both alpha2delta-1-/- male and female mice have reduced Ca2+ influx. Nonetheless, only pancreatic islets from male alpha2delta-1-/- mice show reduced and delayed Ca2+ transients, while islets from female alpha2delta-1-/- mice have unaffected Ca2+ increase, howeverMag. Vincenzo MastroliaIm Titel 2+ hochgestellt und 2 tiefgestelltMedical University of Innsbruck, Dissertation, 2017(VLID)174470

Plastic Pollution: Are Bioplastics the Right Solution?

The adverse effects of the accumulation of plastic on our planet are no longer sustainable; plastic is a major threat to all forms of life in all environments in addition to contributing to global warming. The academic world has been focusing on registering the damages caused by plastic pollution and finding solutions to refrain from and substitute plastic and its usages, which our consumer society is so heavily dependent on. A pathway towards limiting the use of plastic comes from the European Union 2019/904 Directive for limiting the production of single-use and oxo-degradable plastics. Currently, bioplastics are one of the major alternatives in substituting fossil-based plastics, but question remain about its use. as too what extent could bioplastics be a long-term solution to plastic pollution? Is it a misconception to consider bioplastics completely harmless to the environment? This short review article aims to draw attention to the counter effects connected to the limitations and mismanagement of bioplastics through their life cycle by collecting data not published until now. A review of several cradle-to-Grave Life Cycle Assessments has been made to analyse bioplastics from production to end-of-life options. The result produced from this review article shows that bioplastics do not represent a long-term solution to plastic pollution and, on the contrary, may seem to contribute to overall environmental endangerment. The novelty of this work lies in pointing out the misconception of bioplastics’ healthy effects on the environment by thoroughly analysing all environmental impacts of current production and disposal of bioplastics and by providing a more sustainable production of bioplastic through wastewater treatment plants

Loss of a2d-1 calcium channel subunit function increases the susceptibility for diabetes

Reduced pancreatic b-cell function or mass is the critical problem in developing diabetes. Insulin release from b-cells depends on Ca2+ influx through high voltage- gated Ca2+ channels (HVCCs). Ca2+ influx also regulates insulin synthesis and insulin granule priming and contributes to β-cell electrical activity. The HVCCs aremultisubunit protein complexes composed of a pore-forming a1 and auxiliary β and α2δ subunits. α2δ is a key regulator of membrane incorporation and function of HVCCs. Here we show that genetic deletion of α2δ-1, the dominant α 2δ subunit in pancreatic islets, results in glucose intolerance and diabetes without affecting insulin sensitivity. Lack of the α 2δ-1 subunit reduces the Ca2+ currents through all HVCC isoforms expressed in b-cells equally in male and female mice. The reduced Ca2+ influx alters the kinetics and amplitude of the global Ca2+ response to glucose in pancreatic islets and significantly reduces insulin release in both sexes. The progression of diabetes in males is aggravated by a selective loss of b-cell mass, while a stronger basal insulin release alleviates the diabetes symptoms in most α2δ -1 2/2 female mice. Together, these findings demonstrate that the loss of the Ca2+ channel α2β-1 subunit function increases the susceptibility for developing diabetes in a sex-dependent manner

Excitatory and inhibitory synapses show a tight subcellular correlation that weakens over development

Neurons receive correlated levels of excitation and inhibition, a feature that is important for proper brain function. However, how this relationship between excitatory and inhibitory inputs is established during the dynamic period of circuit wiring remains unexplored. Using multiple techniques, including in utero electroporation, electron microscopy, and electrophysiology, we reveal a tight correlation in the distribution of excitatory and inhibitory synapses along the dendrites of developing CA1 hippocampal neurons. This correlation was present within short dendritic stretches (&lt;20 μm) and, surprisingly, was most pronounced during early development, sharply declining with maturity. The tight matching between excitation and inhibition was unexpected, as inhibitory synapses lacked an active zone when formed and exhibited compromised evoked release. We propose that inhibitory synapses form as a stabilizing scaffold to counterbalance growing excitation levels. This relationship diminishes over time, suggesting a critical role for a subcellular balance in early neuronal function and circuit formation.</p

Impulse Conduction Increases Mitochondrial Transport in Adult Mammalian Peripheral Nerves <i>In Vivo</i>

<div><p>Matching energy supply and demand is critical in the bioenergetic homeostasis of all cells. This is a special problem in neurons where high levels of energy expenditure may occur at sites remote from the cell body, given the remarkable length of axons and enormous variability of impulse activity over time. Positioning mitochondria at areas with high energy requirements is an essential solution to this problem, but it is not known how this is related to impulse conduction <i>in vivo</i>. Therefore, to study mitochondrial trafficking along resting and electrically active adult axons <i>in vivo</i>, confocal imaging of saphenous nerves in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons, respectively. We show that low frequency activity induced by electrical stimulation significantly increases anterograde and retrograde mitochondrial traffic in comparison with silent axons. Higher frequency conduction within a physiological range (50 Hz) dramatically further increased anterograde, but not retrograde, mitochondrial traffic, by rapidly increasing the number of mobile mitochondria and gradually increasing their velocity. Similarly, topical application of capsaicin to skin innervated by the saphenous nerve increased mitochondrial traffic in both myelinated and unmyelinated axons. In addition, stationary mitochondria in axons conducting at higher frequency become shorter, thus supplying additional mitochondria to the trafficking population, presumably through enhanced fission. Mitochondria recruited to the mobile population do not accumulate near Nodes of Ranvier, but continue to travel anterogradely. This pattern of mitochondrial redistribution suggests that the peripheral terminals of sensory axons represent sites of particularly high metabolic demand during physiological high frequency conduction. As the majority of mitochondrial biogenesis occurs at the cell body, increased anterograde mitochondrial traffic may represent a mechanism that ensures a uniform increase in mitochondrial density along the length of axons during high impulse load, supporting the increased metabolic demand imposed by sustained conduction.</p></div

In saphenous nerve axons stimulated at high frequency (50 Hz) mitochondria accumulate at the peripheral sensory terminals.

<p>Skin innervated by saphenous nerve was immunohistochemically labelled with VDAC1 (red) and neuron-specific β-III-tubulin (green). In comparison with sham-stimulated animals (A), axons were strongly labelled for VDAC1 in nerves stimulated at 1 Hz (B) and 50 Hz (C). (C′–C′″) Saphenous nerve from a YFP⁺ mouse (shown in low power in C), which expresses YFP (green) in a proportion of fibres, was stimulated with 50 Hz and labelled with VDAC1 (red). (D′–D′″) Saphenous nerve from a YFP⁻ mouse was stimulated with 50 Hz and double labelled with VDAC1 (red) and β-III-tubulin. The two markers were often found to co-localise. (E) Intensity of VDAC1 labelling was significantly higher within cutaneous fibres of saphenous nerve stimulated at 50 Hz (<i>n</i> = 5, <i>p</i><0.05) than in sham-stimulated (<i>n</i> = 3) or fibres stimulated at 1 Hz (<i>n</i> = 3). (F) There was no difference in VDAC1 labelling intensity in DRGs of saphenous nerves between the groups. Scale bars in (A–C) = 100 µm, in (C′–C′″) = 20 µm, in (D′–D′″) = 10 µm.</p