Los adrenoceptores alpha-2 en células cromafines adrenomedulares: papel funcional e implicaciones fisiopatológicas

Chromaffin cells from the adrenal medulla participate in stress responses by releasing catecholamines into the bloodstream. Main control of adrenal catecholamine secretion is exerted both neurally (by the splanchnic nerve fibers) and humorally (by corticosteroids, circulating noradrenaline, etc.). It should be noted, however, that secretory products themselves (catecholamines, ATP, opioids, ascorbic acid, chromogranins) could also influence the secretory response in an autocrine/paracrine manner. This form of control is activity-dependent and can be either inhibitory or excitatory.Among the inhibitory influences, it stands out the one mediated by α2-adrenergic autoreceptors activated by released catecholamines. α2-adrenoceptors areGprotein-coupledreceptors capable to inhibit exocytotic secretion through a direct interaction ofGβγ subunits with voltage-gated Ca2+ channels. Interestingly, upon intense and/or prolonged stimulation, α2-adrenergic receptors become desensitized by the intervention of G protein-coupled receptor kinase 2 (GRK2). In several experimental models of heart failure, there hasbeenreportedthe up-regulationofGRK2 and the loss of functioning of inhibitory α2-adrenoceptors resulting in enhanced release of adrenomedullary catecholamines. Given the importance of circulating catecholamines in the pathophysiology of heart failure, the recovery of α2-adrenergic modulation of the secretory response from chromaffin cells appears as a novel strategy for a better control of the patients with this cardiac disease.Ministerio de Ciencia e Innovación (BFU2011-26253; BFU2015-70067-REDC)Comunidad de Madrid (BRADE-CM S2013/ICE-2958)Sección Deptal. de Farmacología y Toxicología (Veterinaria)Fac. de VeterinariaTRUEpu

Functional remodeling of gap junction-mediated electrical communication between adrenal chromaffin cells in stressed rats.: chromaffin cell coupling in stressed rats

International audienceAn increase in circulating catecholamine levels represents one of the mechanisms whereby organisms cope with stress. In the periphery, catecholamines mainly originate from the sympathoadrenal system. As we reported, in addition to the central control through cholinergic innervation, a local gap junction-delineated route between adrenal chromaffin cells contributes to catecholamine exocytosis. Here, we investigated whether this intercellular communication is modified when the hormonal demand is increased as observed during cold stress. Our results show that in cold exposed rats, gap-junctional communication undergoes a functional plasticity, as evidenced by an increased number of dye-coupled cells. Of a physiological interest is that this upregulation of gap-junctional coupling results in the appearance of a robust electrical coupling between chromaffin cells that allows the transmission of action potentials between coupled cells. This enhancement of gap-junctional communication parallels an increase in expression levels of connexin36 (Cx36) and connexin43 (Cx43) proteins. Both transcriptional and posttranslational mechanisms are involved because Cx36 transcripts are increased in stressed rats and the expression of the scaffolding protein zonula occludens-1, known to interact with both Cx36 and Cx43, is also upregulated. Consistent with an upregulated coupling extent in stressed rats, the cytosolic Ca(2+) concentration rises triggered in a single cell by an iontophoretic application of nicotine occur simultaneously in several neighboring cells. These results describe for the first time a functional plasticity of junctional coupling between adult chromaffin cells that should be crucial for adaptation to stress or sensitization to subsequent stressors

The Adrenal Medulla Modulates Mechanical Allodynia in a Rat Model of Neuropathic Pain

We have investigated whether the stress response mediated by the adrenal medulla in rats subjected to chronic constriction injury of the sciatic nerve (CCI) modulates their nocifensive behavior. Treatment with SK29661 (300 mg/kg; intraperitoneal (I.P.)), a selective inhibitor of phenylethanolamine N-methyltransferase (PNMT) that converts noradrenaline (NA) into adrenaline (A), fully reverted mechanical allodynia in the injured hind paw without affecting mechanical sensitivity in the contralateral paw. The effect was fast and reversible and was associated with a decrease in the A to NA ratio (A/NA) in the adrenal gland and circulating blood, an A/NA that was elevated by CCI. 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (SKF29661) did not affect exocytosis evoked by Ca2+ entry as well as major ionic conductances (voltage-gated Na+, Ca2+, and K+ channels, nicotinic acetylcholine receptors) involved in stimulus-secretion coupling in chromaffin cells, suggesting that it acted by changing the relative content of the two adrenal catecholamines. Denervation of the adrenal medulla by surgical splanchnectomy attenuated mechanical allodynia in neuropathic animals, hence confirming the involvement of the adrenal medulla in the pathophysiology of the CCI model. Inhibition of PNMT appears to be an effective and probably safe way to modulate adrenal medulla activity and, in turn, to alleviate pain secondary to the injury of a peripheral nerve.Universidad Complutense de MadridMinisterio de Ciencia e InnovaciónDepto. de Farmacología y ToxicologíaFac. de VeterinariaTRUEpu

El registro continuo de imagen revela la dinámica de las células madre neurales cerebelosas y el papel de VNUT en la progresión del linaje

Little is known about the intrinsic specification of postnatal cerebellar neural stem cells (NSCs) and to what extent they depend on information from their local niche. Here, we have used an adapted cell preparation of isolated postnatal NSCs and live imaging to demonstrate that cerebellar progenitors maintain their neurogenic nature by displaying hallmarks of NSCs. Furthermore, by using this preparation, all the cell types produced postnatally in the cerebellum, in similar relative proportions to those observed in vivo, can be monitored. The fact that neurogenesis occurs in such organized manner in the absence of signals from the local environment, suggests that cerebellar lineage progression is to an important extent governed by cell-intrinsic or pre-programmed events. Finally, we took advantage of the absence of the niche to assay the influence of the vesicular nucleotide transporter inhibition, which dramatically reduced the number of NSCs in vitro by promoting their progression toward neurogenesis.Comunidad de MadridUniversidad Complutense de MadridFundación Ramón ArecesMinisterio de Educación y Cultura (España)Spanish Ministerio de Ciencia, Innovacio´n y UniversidadesSección Deptal. de Farmacología y Toxicología (Veterinaria)Fac. de VeterinariaTRUEpu

Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy

Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1÷ release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitor-ing that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy.Ion Channel InitiativeMICINNUCM-Banco SantanderComunidad de MadridHealth Research BoardScience Foundation IrelandDepto. de Farmacología y ToxicologíaFac. de VeterinariaTRUEpu

El microARN-22 controla la neurogénesis aberrante y los cambios en la morfología neuronal tras el estado epiléptico

AUTHOR CONTRIBUTIONS EB performed immunohistochemistry, contributed to data analysis and wrote the article. JJ-A performed injections of viral vectors, contributed to data analysis and edited the manuscript. EJ-M and AK performed in situ hybridization. JM and JM-R performed immunohistochemistry. CR performed immunohistochemistry and contributed to data analysis. GL carried out P2X7 immunohistochemistry. LO-O and MA-B performed patch-clamp recordings. SM performed statistical analysis. ND, MF, JJ-A, MM-P, AA and JA edited the manuscript. DO’B provided human brain sample. MD-H edited the manuscript and helped with virus injection. FH provided antibodies and edited the manuscript. DH wrote parts of the manuscript and edited the manuscript. TE performed KA injections and wrote the manuscript.Prolonged seizures (status epilepticus, SE) may drive hippocampal dysfunction and epileptogenesis, at least partly, through an elevation in neurogenesis, dysregulation of migration and aberrant dendritic arborization of newly-formed neurons. MicroRNA-22 was recently found to protect against the development of epileptic foci, but the mechanisms remain incompletely understood. Here, we investigated the contribution of microRNA-22 to SE-induced aberrant adult neurogenesis. SE was induced by intraamygdala microinjection of kainic acid (KA) to model unilateral hippocampal neuropathology in mice. MicroRNA-22 expression was suppressed using specific oligonucleotide inhibitors (antagomir-22) and newly-formed neurons were visualized using the thymidine analog iodo-deoxyuridine (IdU) and a green fluorescent protein (GFP)-expressing retrovirus to visualize the dendritic tree and synaptic spines. Using this approach, we quantified differences in the rate of neurogenesis and migration, the structure of the apical dendritic tree and density and morphology of dendritic spines in newly-formed neurons. SE resulted in an increased rate of hippocampal neurogenesis, including within the undamaged contralateral dentate gyrus (DG). Newly-formed neurons underwent aberrant migration, both within the granule cell layer and into ectopic sites. Inhibition of microRNA-22 exacerbated these changes. The dendritic diameter and the density and average volume of dendritic spines were unaffected by SE, but these parameters were all elevated in mice in which microRNA-22 was suppressed. MicroRNA-22 inhibition also reduced the length and complexity of the dendritic tree, independently of SE. These data indicate that microRNA-22 is an important regulator of morphogenesis of newly-formed neurons in adults and plays a role in supressing aberrant neurogenesis associated with SE.Science Foundation Ireland (13/SIRG/2098; 17/CDA/4708; 16/TIDA/4059; 13/SIRG/2114; 17/TIDA/5002; 13/IA/1891; 16/RC/3948)European Regional Development Fund and by FutureNeuro Industry partners) from the Health Research Board (HRA-POR-2015-1243)European Union Seventh Framework Programme (FP7/2007–2013)H2020 Marie Skłowdowksa-Curie Actions(grants nº 753527; 766124)Comunidad de Madrid (S2013/ICE-2958)Fundación Ramón ArecesFundación La CaixaMinisterio de Ciencia y Universidades (BFU2014-53654-P)Sección Deptal. de Farmacología y Toxicología (Veterinaria)Fac. de VeterinariaTRUEpu

Loose coupling between calcium channels and sites of exocytosis in chromaffin cells

Calcium microdomains generated by tight clusters of calcium channels regulate fusion of small vesicles at the synaptic terminal and have also been suggested to trigger exocytosis of large dense-core vesicles from neuroendocrine cells. To test this idea, we have compared sites of exocytosis and the spatial distribution of calcium channels in chromaffin cells. Fusion of individual vesicles was visualized using interference reflection microscopy and the submembranous calcium signal was assessed using total internal reflection fluorescence microscopy. Depolarization triggered a burst of exocytosis from up to seven sites in a membrane area of 11 μm2, but these sites did not colocalize with calcium microdomains. Instead, calcium influx occurred in large patches (averaging 34 μm2) containing a mixture of P/Q- and N-type channels. About 20% of fusion events occurred outside calcium channel patches. Further, the delay between the onset of stimulation and a burst of exocytosis was prolonged for several seconds by increasing the concentration of the slow calcium chelator EGTA from 1.5 to 5 mm. These results demonstrate that while calcium channels and release sites tend to congregate in specialized regions of the surface membrane, these have dimensions of several micrometres. The dominant calcium signal regulating release in chromaffin cells is generated by the cooperative action of many channels operating over distances of many micrometres rather than discrete clusters of calcium channels generating localized microdomains