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Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla
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Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla Christian Schnell a,c,1 , Mahmoud Negm b,1 , Johannes Driehaus b , Anja Scheller d , Swen Hülsmann a,b,∗ a
DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany Clinic for Anesthesiology, University Hospital Göttingen, 37099 Göttingen, Germany c Divisions of Pathophysiology & Repair and Neuroscience, School of Biosciences, Cardiff University, Cardiff, United Kingdom d Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, Homburg, Germany b
a r t i c l e
i n f o
Article history: Received 26 July 2015 Received in revised form 14 October 2015 Accepted 15 October 2015 Available online xxx Keywords: Glia Catecholaminergic signaling Neuronal control of breathing
a b s t r a c t The neuronal activity in the respiratory network of the ventrolateral medulla strongly depends on a variety of different neuromodulators. Since the respiratory activity generated by neurons in the pre-Bötzinger complex (preBötC) is stabilized by astrocytes, we investigated potential effects of the neuromodulator norepinephrine (NE) on the astrocytic calcium signaling in the ventral respiratory group. In acutely isolated brainstem slices from wild type mice (postnatal day 1–10) we performed calcium imaging experiments using Oregon Green 488 BAPTA-1 AM as a calcium indicator dye. Astrocytes in the preBötC, which were identified by their unique intracellular calcium rise after the reduction of the extracellular K+ concentration, showed calcium rises in response to norepinephrine. These calcium signals persisted after blockade of neuronal activity by tetrodotoxin (TTX) indicating that they were independent of neuronal activity. Furthermore, application of the endoplasmic reticulum calcium pump blocker cyclopiazonic acid (CPA) diminished norepinephrine-induced calcium signals. This results could be confirmed using transgenic mice with astrocyte specific expression of GCaMP3. Thus, norepinephrine might, apart from acting directly on neurons, influence and modulate respiratory network activity via the modulation of astroglial calcium signaling. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the ventrolateral medulla neurons for respiratory and cardiac control are intermingled and surrounded by astrocytes that stabilize autonomous activity of the network (Hülsmann et al., 2000; Grass et al., 2004; Schnell et al., 2011; Oku et al., 2015). Not only have these cells been shown to provide supportive functions for this neuronal activity, e.g., by mediating reuptake of neurotransmitters (Gomeza et al., 2003; Szöke et al., 2006), but they also have been shown to respond to various neuromodulators by an increase of their intracellular calcium concentration (Funk et al., 2008; Härtel et al., 2009; Huxtable et al., 2010). Like other neuromodulators norepinephrine (NE) has been shown to stimulate the output of the respiratory network activity (Errchidi et al., 1991; Al-Zubaidy et al., 1996; Viemari and Ramirez, 2006; Funk et al., 2011). NE is released
∗ Corresponding author. Clinic for Anesthesiology, University Hospital Göttingen, 37099 Göttingen, Germany. Fax: +49 551 399676. E-mail address: [email protected] (S. Hülsmann). 1 Equally contributed.
in the respiratory network from pontine and medullary neurons (Robertson et al., 2013) and leading to a stimulation of ventilation (Bianchi et al., 1995; Hilaire et al., 2004). Moreover, medullary NE levels are reduced in mouse models for the RETT syndrome (Viemari et al., 2005). To understand the role of astrocytes in NEsignaling we aimed to investigate NE-effects, which might involve astrocytes. Here we tested if NE exerts an effect on astrocytes and their main signaling mechanism of calcium signaling. 2. Materials and methods 2.1. Breeding of mice Animals were hold and bred in the animal facilities of the University Hospital Göttingen in accordance with guidelines of the German Physiological Society as well as the regulations of the State of Lower Saxony Institutional registration (T19/08), and Saarland and the Federal Republic of Germany (TierSchG). Procedures were approved by the authorities of Lower Saxony State Office for Consumer Protection and Food Safety; 33.12-42502-04-14/1524 and Saarland (State Office for Consumer Protection; 72/2010).
http://dx.doi.org/10.1016/j.resp.2015.10.008 1569-9048/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008
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Experiments were performed on acute brain slice preparations of NMRI (naval medical research institute), transgenic hGFAP-mRFP1 (Hirrlinger et al., 2005) and Glast-GCaMP3 mice (Mori et al., 2006; Paukert et al., 2014). 2.2. Induction of GCaMP3 expression in astrocytes Glast-creERT2 mice (Slc1a3tm1(cre/ERT2)Mgoe ; Mori et al., 2006) and R26-lsl-GCaMP3 (Paukert et al., 2014) were crossbred to receive double transgenic offsprings. For induction of GCaMP3 expression in newborn offsprings, lactating mice were intraperionetally injected with tamoxifen (10 mg/ml solved in corn oil, 100 mg/kg body weight) once a day for 1–4 consecutive days on day 0–3 after birth. After the last tamoxifen injection we waited a minimum of 2 days for allowing sufficient expression of GCaMP3. 2.3. Slice preparations Acute transversal brainstem slices were prepared as described previously (Härtel et al., 2007; Winter et al., 2009; Schnell et al., 2011). Animals were decapitated under diethyl-ether anesthesia, and brainstems were isolated and placed in ice-cooled, carbogensaturated (95% O2 , 5% CO2 ) artificial cerebrospinal fluid (aCSF) containing 118 mM NaCl, 3 KCl, 1.5 mM CaCl2 , 1 mM MgCl2 , 1 mM NaH2 PO4, 25 mM NaHCO3 , and 30 mM d-glucose; pH 7.4. The isolated brainstem was glued with cyanoacryl glue (Loctite Deutschland GmbH) to an agar block and mounted in a vibroslicer (VT 1000S, Leica). The location of the preBötC was judged based on the anatomy of inferior olive and 4th ventricle (see: Winter et al., 2009; Schnell et al., 2011). Before staining with Oregon Green BAPTA 1-AM (OGB; see below) slices were kept in oxygenated ACSF at room temperature for at least 30 min. For imaging experiments, slices were transferred to the recording chamber and were kept submerged by a nylon fiber grid and continuously perfused with aCSF at a flow rate of 5–10 ml/min. For OGB calcium imaging experiments 200–300 m slices containing the pre-Bötzinger complex (preBötC) were cut from P1–P10 mice. Slices made from GCaMP3 expressing mice (P3–P11) were 650 m thick. 2.4. Epifluorescence calcium imaging
Fig. 1. Analysis of norepinephrine (NE)-induced calcium signals in astrocytes of the ventrolateral medulla. A: Oregon-Green-BAPTA1-AM labeling of the tissue. B: Identification of astrocytes was performed via application of an extracellular solution with a low (0.2 mM) potassium concentration. The F/F0 image identifies the position of astrocytes by the calcium rise during application of 0.2 mM K+ . C: Original traces from the cells indicated in panel A,B. NE (10 M) is applied as indicated before and during the application of TTX. Glutamate (1 mM) was applied to prove the viability of the cells. D: Application of CPA diminished the response of NE. E: Significant reduction of calcium signals induced by NE in the presence of the ␣1-adrenoreceptor antagonist prazosin (50 M).
Cells were loaded with the calcium dye Oregon Green BAPTA 1AM by multi-cell bolus loading (Zhao et al., 2006) and fluorescence changes were recorded using upright fluorescence microscopy (Axioscope FS1, Zeiss) though a 40 × 0.8 NA water immersion objective (Zeiss) with a cooled CCD camera (SensiCam; PCO, Kelheim, Germany). A monochromator (Polychrome, T.I.L.L. Photonics, München, Germany) was used to generate excitation (494 nm). For wavelength control and image acquisition Imaging Workbench 6 (Indec Biosystems, Mt. View, USA) was used and images were captured at 1 Hz. During epifluorescence imaging, astrocytes were identified by their characteristic calcium response induced by lowering the extracellular potassium concentration from 3 mM to 0.2 mM. This procedure was also used to identify astrocytes in hGFAP-mRFP1 mice (Härtel et al., 2009; Hirrlinger et al., 2005) and has been shown to be a reliable method to identify astrocytes in brain slices (Dallwig et al., 2000; Dallwig and Deitmer, 2002; Härtel et al., 2007). 2.5. 2-photon excitation microscopy GCaMP3 fluorescence was imaged through a 20 × 1.0 NA water immersion objective (Zeiss) with non-descanned detection by
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008
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Fig. 2. NE-induced calcium signals in transgenic mice expressing the genetically encoded calcium indicator GCaMP3 in astrocytes. A: 2-Photon micrograph of the GCaMP3 expression in the ventrolateral medulla of a P3 mouse. B: F/F0 image during the application of NE (200 M) and C: Calcium signal of eight astrocytes in response to NE-application.
GaAsP Photomultipliers (Hamamatsu) using a 2-photon excitation microscopy unit (TriMScope 1, LaVision BioTec) connected to an Axioscope FS2 (Zeiss). 2-Photon excitation was achieved with a Ti:Sapphire Laser (SpectraPhysics MaiTai BB) at 850 nm. Fluorescence signals of GCaMP3-expressing astrocytes were detected through a 531/40 nm band pass emission filter (AHF Analysentechnik AG). 2.6. Drugs Electrolytes for aCSF (see above) and tamoxifen were purchased from Sigma–Aldrich (Taufkirchen, Germany) and Merck chemicals (Darmstadt, Germany). Agonists and antagonists were brought from Tocris Bioscience (Bristol, UK) and bath applied at concentrations indicated in the text. 2.7. Data analysis Data are expressed as mean ± SEM. Statistical comparison was performed with the SigmaPlot software (Systat Software, Inc.). Statistical significance (paired t-tests) was assumed if p < 0.05. 3. Results 3.1. Astrocytic norepinephrine induced calcium responses in the ventrolateral medulla In a first set of experiments, we tested whether norepinephrine (NE) has a direct effect on astrocytes in the ventrolateral medulla. Therefore, we applied NE (10 M) in the presence and absence of tetrodotoxin (TTX) that blocks neuronal action potentials. There was no significant change of the amplitude of the NE-induced cal-
cium rise in astrocytes (0.514 ± 0.216 (F/F0 ) in control (CTRL) and 0.521 ± 0.216 (F/F0 ) in TTX; n.s.; n = 6 slices / 3 animals; Fig. 1C). Astrocytes often respond to neuromodulators by a release of calcium from intracellular stores (Härtel et al., 2009). To test if the NE-induced response is mediated by an intracellular calcium release, we applied cyclopiazonic acid (CPA), a blocker of the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) that depletes intracellular calcium stores (Le Pichon and Chesler, 2014). In the presence of CPA, the amplitude of the calcium response in astrocytes was significantly diminished (CTRL 0.453 ± 0.191 (F/F0 ) vs. CPA 0.026 ± 0.0360 (F/F0 ); p = 0.003; n = 7 slices / 4 animals; Fig. 1D) indicating that intracellular calcium release is the major source of the astrocytic NE-induced calcium signal. Different adrenergic receptors have been described in astrocytes of the brainstem including the ␣1-adrenergic receptor that is known to cause release of calcium from intracellular stores. Indeed application of the ␣1-adrenregic receptor blocker prazosin (50 M; (Nilsson et al., 1992)) significantly reduced the NE-induced calcium response (0.283 ± 0.111 (F/F0 ) CTRL vs. 0.092 ± 0.130 (F/F0 ) in prazosin; P = 0.008; n = 6 slices / 3 animals; Fig. 1E) indicating that NE-acts via G protein coupled Phospholipase C-Inositol trisphosphate cascade (Gq-PLC-IP3). Imaging using camera based detection and AM-ester dyes bears the risk of contamination of light from neighboring cells (see (Oku et al., 2015) this issue). To overcome this problem we confirmed NE-induced calcium signals in astrocytes that express the genetically encoded calcium sensor GCaMP3 controlled by the glutamate/aspartate transporter locus (Glast, EAAT1); n = 6 slices / 6 animals. After application of NE (50– 200 M) astrocytes responded with a typical oscillatory calcium signal (Fig. 2), that was earlier described for type 1 metabotropic glutamate receptor agonists (Schnell et al., 2011).
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Fig. 3. A: Schematic drawing of astrocytic norepinephrine signaling. B: Table of the expression of key players of the adrenergic signaling in the medulla based on cell type-specific next generation sequencing (Schnell et al., 2015).
4. Discussion Norepinephrine (NE) increases intracellular calcium in the preBötC astrocytes. This astrocytic calcium signal might mediate an indirect contribution to the well-known stimulatory NE effect on the respiratory network activity as suggested for metabotropic glutamate receptors (Schnell et al., 2011). Norepinephrine acts via ␣1-adrenoreceptors that release calcium from intracellular stores via the Gq-PLC-IP3 pathway (Fig. 3). Indeed, ␣1-adrenoreceptors were identified on Bergmann-Glia (Kirischuk et al., 1996) or astrocytes in the hippocampus (Duffy and MacVicar, 1995). These receptors were also suggested to be responsible for the calcium rise in the preBötC (Ruangkittisakul et al., 2009). Our data supports this concept and strengthen the hypothesis by showing that the calcium signal induced by NE does not result from an optical signal contamination from neighboring neuronal processes (Fig. 2). Moreover, the persistence of the NE-induced calcium signal in the presence of TTX excludes the possibility that the calcium rise in the astrocyte is mediated indirectly via activation of NE-receptors expressing neurons that release e.g., glutamate. Next generation sequencing from brainstem astroglial mRNA (Schnell et al., 2015) showed additional astrocytic expression of ␣2- and 1-adrenoreceptors (Fig. 3). ␣2- and 1-adrenoreceptors, which antagonistically regulate cAMP levels via Gi/o and GS , respectively, might have contributed to the remaining calcium signal after blockade of ␣1-adrenoreceptors by prazosin (Fig. 1E). Although astrocytic calcium levels potentially can be modulated by the exchange protein directly activated by
cAMP (EPAC) pathway (Di Cesare et al., 2006), the current data do not suggest a major contribution of the EPAC signaling pathway. 4.1. Technical consideration To induced GCaMP3 expression in astrocytes, tamoxifen was administered to lactating mice in order to induce GCaMP3 expression in neonatal mice (Leone et al., 2003; Hirrlinger et al., 2006). The tamoxifen-inducible Cre/loxP gene recombination system is frequently used to allow temporal control of gene expression (Jahn et al., 2015) and increases the cell-type specificity of the expression of genetically encoded calcium sensors in astrocytes (Paukert et al., 2014). However, there are reports of unspecific side effects of tamoxifen that have been reported e.g., in cell culture. With respect to astroglial calcium signaling, tamoxifen can influence and interfere with glutamate transporters in astrocytes (Sato et al., 2008; Karki et al., 2013), via upregulation of expression (Karki et al., 2013) or via decrease of effective glutamate transport (Sato et al., 2008). Indeed it has to be assumed that during the induction phase, brain tissue levels of tamoxifen reach concentrations comparable with these culture experiments (Iusuf et al., 2011). More important for our NE-experiments, tamoxifen like other estrogen receptor agonists, can further interfere with the astroglial intracellular calcium signaling cascade by inhibition of the PKC (O’Brian et al., 1985). Thus, we cannot exclude that glutamate signaling was temporarily altered. However, it has been shown in other studies that tamoxifen did not mediate any persistent effect on glutamate transport
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008
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(measures by means of glutamate receptor currents) that could not be explained by the induction of recombination (Saab et al., 2012), making an acute effect that impairs our interpretation of the experiments unlikely. Indeed, we did not observe an increase of spontaneous calcium oscillation in astrocyte in mice with GCaMP3 expression in comparison to the current experiments with Oregon green calcium indicator nor to our earlier data (Härtel et al., 2009; Schnell et al., 2011), suggesting that glutamate levels in the slice preparation of GCaMP3-expressing mice are normal. However, in the current paper GCaMP3 expressing astrocytes (Paukert et al., 2014) were used to confirm that NE-induced calcium signals results indeed from astrocytes and not from the neighboring neuropil (Oku et al., 2015). This aim could be achieved, and we are confident not to have overlooked a relevant factor. 4.2. Implications for respiratory physiology and pathophysiology Since norepinephrine has recently been shown to be a major factor controlling the responsiveness of astrocytes to neuronal network activity (Paukert et al., 2014) we suggest that, when interpreting the role of norepinephrine in healthy and disease states of the respiratory network, astroglial calcium signaling should be taken into consideration. This might be particularly relevant in mouse models of the RETT syndrome, in which the respiratory phenotype depends on Mecp2 levels in astrocytes (Lioy et al., 2011). Thus, the significant changes of norepinephrine concentration that occurs in Mecp2-deficient mice (Viemari et al., 2005) can be expected to alter the network activity both directly and indirectly by changing astroglial calcium signaling and potentially also the levels of glio-transmitters like ATP or glutamate (Volterra et al., 2014). Acknowledgements We thank A.-A. Grützner for technical support. The authors are grateful to D. Rhode and T. Vogelgesang for animal husbandry and tamoxifen treatments (University of Saarland). The work was supported by the “Deutsche Forschungsgemeinschaft” through DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) and personal grant to S.H. (Hu797/5-1). The authors are grateful to M. Götz (Munich) and D. Bergles (Baltimore) for providing mice that founded the breeding of the transgenic mice used for our experiments. References Al-Zubaidy, Z.A., Erickson, R.L., Greer, J.J., 1996. Serotonergic and noradrenergic effects on respiratory neural discharge in the medullary slice preparation of neonatal rats. Pflugers Arch. 431, 942–949. Bianchi, A.L., Denavit-Saubie, M., Champagnat, J., 1995. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75, 1–45. Dallwig, R., Deitmer, J.W., 2002. Cell-type specific calcium responses in acute rat hippocampal slices. J. Neurosci. Methods 116, 77–87. Dallwig, R., Vitten, H., Deitmer, J.W., 2000. A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28, 247–259. Di Cesare, A., Del Piccolo, P., Zacchetti, D., Grohovaz, F., 2006. EP2 receptor stimulation promotes calcium responses in astrocytes via activation of the adenylyl cyclase pathway. Cell. Mol. Life Sci. CMLS 63, 2546–2553. Duffy, S., MacVicar, B.A., 1995. Adrenergic calcium signaling in astrocyte networks within the hippocampal slice. J. Neurosci. 15, 5535–5550. Errchidi, S., Monteau, R., Hilaire, G., 1991. Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study. J.Physiol. 443, 477–498. Funk, G.D., Huxtable, A.G., Lorier, A.R., 2008. ATP in central respiratory control: a three-part signaling system. Respir. Physiol. Neurobiol. 164, 131–142. Funk, G.D., Zwicker, J.D., Selvaratnam, R., Robinson, D.M., 2011. Noradrenergic modulation of hypoglossal motoneuron excitability: developmental and putative state-dependent mechanisms. Arch. Ital. Biol. 149, 426–453. Gomeza, J., Hülsmann, S., Ohno, K., Eulenburg, V., Szoke, K., Richter, D., Betz, H., 2003. Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition. Neuron 40, 785–796.
5
Grass, D., Pawlowski, P.G., Hirrlinger, J., Papadopoulos, N., Richter, D.W., Kirchhoff, F., Hülsmann, S., 2004. Diversity of functional astroglial properties in the respiratory network. J. Neurosci. 24, 1358–1365. Härtel, K., Schnell, C., Hülsmann, S., 2009. Astrocytic calcium signals induced by neuromodulators via functional metabotropic receptors in the ventral respiratory group of neonatal mice. Glia 57, 815–827. Härtel, K., Singaravelu, K., Kaiser, M., Neusch, C., Hülsmann, S., Deitmer, J.W., 2007. Calcium influx mediated by the inwardly rectifying K+ channel Kir4.1 (KCNJ10) at low external K+ concentration. Cell Calcium 42, 271–280. Hilaire, G., Viemari, J.C., Coulon, P., Simonneau, M., Bevengut, M., 2004. Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents. Respir. Physiol. Neurobiol. 143, 187–197. Hirrlinger, P.G., Scheller, A., Braun, C., Hirrlinger, J., Kirchhoff, F., 2006. Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2. Glia 54, 11–20. Hirrlinger, P.G., Scheller, A., Braun, C., Quintela-Schneider, M., Fuss, B., Hirrlinger, J., Kirchhoff, F., 2005. Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice. Mol. Cell Neurosci. 30, 291–303. Hülsmann, S., Oku, Y., Zhang, W., Richter, D.W., 2000. Metabolic coupling between glia and neurons is necessary for maintaining respiratory activity in transverse medullary slices of neonatal mouse. Eur. J. Neurosci. 12, 856–862. Huxtable, A.G., Zwicker, J.D., Alvares, T.S., Ruangkittisakul, A., Fang, X., Hahn, L.B., Posse de Chaves, E., Baker, G.B., Ballanyi, K., Funk, G.D., 2010. Glia contribute to the purinergic modulation of inspiratory rhythm-generating networks. J. Neurosci. 30, 3947–3958. Iusuf, D., Teunissen, S.F., Wagenaar, E., Rosing, H., Beijnen, J.H., Schinkel, A.H., 2011. P-glycoprotein (ABCB1) transports the primary active tamoxifen metabolites endoxifen and 4-hydroxytamoxifen and restricts their brain penetration. J. Pharmacol. Exp. Ther. 337, 710–717. Jahn, H.M., Scheller, A., Kirchhoff, F., 2015. Genetic control of astrocyte function in neural circuits. Front. Cell. Neurosci. 9. Karki, P., Webb, A., Smith, K., Lee, K., Son, D.-S., Aschner, M., Lee, E., 2013. cAMP response element-binding protein (CREB) and nuclear factor B mediate the tamoxifen-induced up-regulation of glutamate transporter 1 (GLT-1) in rat astrocytes. J. Biol. Chem. 288, 28975–28986. Kirischuk, S., Tuschick, S., Verkhratsky, A., Kettenmann, H., 1996. Calcium signalling in mouse Bergmann glial cells mediated by alpha1-adrenoreceptors and H1 histamine receptors. Eur. J. Neurosci. 8, 1198–1208. Le Pichon, C.E., Chesler, A.T., 2014. The functional and anatomical dissection of somatosensory subpopulations using mouse genetics. Front. Neuroanat., 8. Leone, D.P., Genoud, St., Aanasoski, S., Grausenburger, R., Berger, P., Metzger, D., Macklin, W.B., Chambon, P., Suter, U., 2003. Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells. Mol. Cell. Neurosci. 22, 430–440. Lioy, D.T., Garg, S.K., Monaghan, C.E., Raber, J., Foust, K.D., Kaspar, B.K., Hirrlinger, P.G., Kirchhoff, F., Bissonnette, J.M., Ballas, N., Mandel, G., 2011. A role for glia in the progression of Rett/’s syndrome. Nature advance online publication. Mori, T., Tanaka, K., Buffo, A., Wurst, W., Kuhn, R., Götz, M., 2006. Inducible gene deletion in astroglia and radial glia–a valuable tool for functional and lineage analysis. Glia 54, 21–34. Nilsson, M., Hansson, E., Ronnback, L., 1992. Agonist-evoked Ca2+ transients in primary astroglial cultures—modulatory effects of valproic acid. Glia 5, 201–209. O’Brian, C.A., Liskamp, R.M., Solomon, D.H., Weinstein, I.B., 1985. Inhibition of Protein Kinase C by Tamoxifen. Cancer Res. 45, 2462–2465. Oku Y, Fresemann J, Miwakeichi F, Hülsmann S. (2015) Respiratory calcium fluctuations in low-frequency oscillating astrocytes in the pre-Botzinger complex. Respir Physiol Neurobiol. Paukert, M., Agarwal, A., Cha, J., Doze, V.A., Kang, J.U., Bergles, D.E., 2014. Norepinephrine controls astroglial responsiveness to local circuit activity. Neuron 82, 1263–1270. Robertson, S.D., Plummer, N.W., de Marchena, J., Jensen, P., 2013. Developmental origins of central norepinephrine neuron diversity. Nat. Neurosci. 16, 1016–1023. Ruangkittisakul, A., Okada, Y., Oku, Y., Koshiya, N., Ballanyi, K., 2009. Fluorescence imaging of active respiratory networks. Respir. Physiol. Neurobiol. 168, 26–38. ˇ Saab, A.S., Neumeyer, A., Jahn, H.M., Cupido, A., Simek, A.A.M., Boele, H.-J., Scheller, A., Le Meur, K., Götz, M., Monyer, H., Sprengel, R., Rubio, M.E., Deitmer, J.W., De Zeeuw, C.I., Kirchhoff, F., 2012. Bergmann Glial AMPA receptors are required for fine motor coordination. Science 337, 749–753. Sato, K., Saito, Y., Oka, J.-, i, O., hwada, T., Nakazawa, K., 2008. Effects of Tamoxifen on L-Glutamate Transporters of Astrocytes. J. Pharmacol. Sci. 107, 226–230. Schnell, C., Fresemann, J., Hülsmann, S., 2011. Determinants of functional coupling between astrocytes and respiratory neurons in the pre-Botzinger complex. PLoS One 6, e26309. Schnell, C., Shahmoradi, A., Wichert, S.P., Mayerl, S., Hagos, Y., Heuer, H., Rossner, M.J., Hülsmann, S., 2015. The multispecific thyroid hormone transporter OATP1C1 mediates cell-specific sulforhodamine 101-labeling of hippocampal astrocytes. Brain Struct. Funct. 220, 193–203. Szöke, K., Härtel, K., Grass, D., Hirrlinger, P.G., Hirrlinger, J., Hülsmann, S., 2006. Glycine transporter 1 expression in the ventral respiratory group is restricted to protoplasmic astrocytes. Brain Res. 1119, 182–189. Viemari, J.C., Ramirez, J.M., 2006. Norepinephrine differentially modulates different types of respiratory pacemaker and nonpacemaker neurons. J. Neurophysiol. 95, 2070–2082.
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Viemari, J.C., Roux, J.C., Tryba, A.K., Saywell, V., Burnet, H., Pena, F., Zanella, S., Bevengut, M., Barthelemy-Requin, M., Herzing, L.B., Moncla, A., Mancini, J., Ramirez, J.M., Villard, L., Hilaire, G., 2005. Mecp2 deficiency disrupts norepinephrine and respiratory systems in mice. J. Neurosci. 25, 11521–11530. Volterra, A., Liaudet, N., Savtchouk, I., 2014. Astrocyte Ca2+ signalling: an unexpected complexity. Nat. Rev. Neurosci. 15, 327–335.
Winter, S.M., Fresemann, J., Schnell, C., Oku, Y., Hirrlinger, J., Hülsmann, S., 2009. Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices. Pflugers Arch. 458, 459–469. Zhao, M.G., Hülsmann, S., Winter, S.M., Dutschmann, M., Richter, D.W., 2006. Calcium-regulated potassium currents secure respiratory rhythm generation after loss of glycinergic inhibition. Eur. J. Neurosci. 24, 145–154.
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Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla Christian Schnell a,c,1 , Mahmoud Negm b,1 , Johannes Driehaus b , Anja Scheller d , Swen Hülsmann a,b,∗ a
DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany Clinic for Anesthesiology, University Hospital Göttingen, 37099 Göttingen, Germany c Divisions of Pathophysiology & Repair and Neuroscience, School of Biosciences, Cardiff University, Cardiff, United Kingdom d Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, Homburg, Germany b
a r t i c l e
i n f o
Article history: Received 26 July 2015 Received in revised form 14 October 2015 Accepted 15 October 2015 Available online xxx Keywords: Glia Catecholaminergic signaling Neuronal control of breathing
a b s t r a c t The neuronal activity in the respiratory network of the ventrolateral medulla strongly depends on a variety of different neuromodulators. Since the respiratory activity generated by neurons in the pre-Bötzinger complex (preBötC) is stabilized by astrocytes, we investigated potential effects of the neuromodulator norepinephrine (NE) on the astrocytic calcium signaling in the ventral respiratory group. In acutely isolated brainstem slices from wild type mice (postnatal day 1–10) we performed calcium imaging experiments using Oregon Green 488 BAPTA-1 AM as a calcium indicator dye. Astrocytes in the preBötC, which were identified by their unique intracellular calcium rise after the reduction of the extracellular K+ concentration, showed calcium rises in response to norepinephrine. These calcium signals persisted after blockade of neuronal activity by tetrodotoxin (TTX) indicating that they were independent of neuronal activity. Furthermore, application of the endoplasmic reticulum calcium pump blocker cyclopiazonic acid (CPA) diminished norepinephrine-induced calcium signals. This results could be confirmed using transgenic mice with astrocyte specific expression of GCaMP3. Thus, norepinephrine might, apart from acting directly on neurons, influence and modulate respiratory network activity via the modulation of astroglial calcium signaling. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the ventrolateral medulla neurons for respiratory and cardiac control are intermingled and surrounded by astrocytes that stabilize autonomous activity of the network (Hülsmann et al., 2000; Grass et al., 2004; Schnell et al., 2011; Oku et al., 2015). Not only have these cells been shown to provide supportive functions for this neuronal activity, e.g., by mediating reuptake of neurotransmitters (Gomeza et al., 2003; Szöke et al., 2006), but they also have been shown to respond to various neuromodulators by an increase of their intracellular calcium concentration (Funk et al., 2008; Härtel et al., 2009; Huxtable et al., 2010). Like other neuromodulators norepinephrine (NE) has been shown to stimulate the output of the respiratory network activity (Errchidi et al., 1991; Al-Zubaidy et al., 1996; Viemari and Ramirez, 2006; Funk et al., 2011). NE is released
∗ Corresponding author. Clinic for Anesthesiology, University Hospital Göttingen, 37099 Göttingen, Germany. Fax: +49 551 399676. E-mail address: [email protected] (S. Hülsmann). 1 Equally contributed.
in the respiratory network from pontine and medullary neurons (Robertson et al., 2013) and leading to a stimulation of ventilation (Bianchi et al., 1995; Hilaire et al., 2004). Moreover, medullary NE levels are reduced in mouse models for the RETT syndrome (Viemari et al., 2005). To understand the role of astrocytes in NEsignaling we aimed to investigate NE-effects, which might involve astrocytes. Here we tested if NE exerts an effect on astrocytes and their main signaling mechanism of calcium signaling. 2. Materials and methods 2.1. Breeding of mice Animals were hold and bred in the animal facilities of the University Hospital Göttingen in accordance with guidelines of the German Physiological Society as well as the regulations of the State of Lower Saxony Institutional registration (T19/08), and Saarland and the Federal Republic of Germany (TierSchG). Procedures were approved by the authorities of Lower Saxony State Office for Consumer Protection and Food Safety; 33.12-42502-04-14/1524 and Saarland (State Office for Consumer Protection; 72/2010).
http://dx.doi.org/10.1016/j.resp.2015.10.008 1569-9048/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008
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Experiments were performed on acute brain slice preparations of NMRI (naval medical research institute), transgenic hGFAP-mRFP1 (Hirrlinger et al., 2005) and Glast-GCaMP3 mice (Mori et al., 2006; Paukert et al., 2014). 2.2. Induction of GCaMP3 expression in astrocytes Glast-creERT2 mice (Slc1a3tm1(cre/ERT2)Mgoe ; Mori et al., 2006) and R26-lsl-GCaMP3 (Paukert et al., 2014) were crossbred to receive double transgenic offsprings. For induction of GCaMP3 expression in newborn offsprings, lactating mice were intraperionetally injected with tamoxifen (10 mg/ml solved in corn oil, 100 mg/kg body weight) once a day for 1–4 consecutive days on day 0–3 after birth. After the last tamoxifen injection we waited a minimum of 2 days for allowing sufficient expression of GCaMP3. 2.3. Slice preparations Acute transversal brainstem slices were prepared as described previously (Härtel et al., 2007; Winter et al., 2009; Schnell et al., 2011). Animals were decapitated under diethyl-ether anesthesia, and brainstems were isolated and placed in ice-cooled, carbogensaturated (95% O2 , 5% CO2 ) artificial cerebrospinal fluid (aCSF) containing 118 mM NaCl, 3 KCl, 1.5 mM CaCl2 , 1 mM MgCl2 , 1 mM NaH2 PO4, 25 mM NaHCO3 , and 30 mM d-glucose; pH 7.4. The isolated brainstem was glued with cyanoacryl glue (Loctite Deutschland GmbH) to an agar block and mounted in a vibroslicer (VT 1000S, Leica). The location of the preBötC was judged based on the anatomy of inferior olive and 4th ventricle (see: Winter et al., 2009; Schnell et al., 2011). Before staining with Oregon Green BAPTA 1-AM (OGB; see below) slices were kept in oxygenated ACSF at room temperature for at least 30 min. For imaging experiments, slices were transferred to the recording chamber and were kept submerged by a nylon fiber grid and continuously perfused with aCSF at a flow rate of 5–10 ml/min. For OGB calcium imaging experiments 200–300 m slices containing the pre-Bötzinger complex (preBötC) were cut from P1–P10 mice. Slices made from GCaMP3 expressing mice (P3–P11) were 650 m thick. 2.4. Epifluorescence calcium imaging
Fig. 1. Analysis of norepinephrine (NE)-induced calcium signals in astrocytes of the ventrolateral medulla. A: Oregon-Green-BAPTA1-AM labeling of the tissue. B: Identification of astrocytes was performed via application of an extracellular solution with a low (0.2 mM) potassium concentration. The F/F0 image identifies the position of astrocytes by the calcium rise during application of 0.2 mM K+ . C: Original traces from the cells indicated in panel A,B. NE (10 M) is applied as indicated before and during the application of TTX. Glutamate (1 mM) was applied to prove the viability of the cells. D: Application of CPA diminished the response of NE. E: Significant reduction of calcium signals induced by NE in the presence of the ␣1-adrenoreceptor antagonist prazosin (50 M).
Cells were loaded with the calcium dye Oregon Green BAPTA 1AM by multi-cell bolus loading (Zhao et al., 2006) and fluorescence changes were recorded using upright fluorescence microscopy (Axioscope FS1, Zeiss) though a 40 × 0.8 NA water immersion objective (Zeiss) with a cooled CCD camera (SensiCam; PCO, Kelheim, Germany). A monochromator (Polychrome, T.I.L.L. Photonics, München, Germany) was used to generate excitation (494 nm). For wavelength control and image acquisition Imaging Workbench 6 (Indec Biosystems, Mt. View, USA) was used and images were captured at 1 Hz. During epifluorescence imaging, astrocytes were identified by their characteristic calcium response induced by lowering the extracellular potassium concentration from 3 mM to 0.2 mM. This procedure was also used to identify astrocytes in hGFAP-mRFP1 mice (Härtel et al., 2009; Hirrlinger et al., 2005) and has been shown to be a reliable method to identify astrocytes in brain slices (Dallwig et al., 2000; Dallwig and Deitmer, 2002; Härtel et al., 2007). 2.5. 2-photon excitation microscopy GCaMP3 fluorescence was imaged through a 20 × 1.0 NA water immersion objective (Zeiss) with non-descanned detection by
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008
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Fig. 2. NE-induced calcium signals in transgenic mice expressing the genetically encoded calcium indicator GCaMP3 in astrocytes. A: 2-Photon micrograph of the GCaMP3 expression in the ventrolateral medulla of a P3 mouse. B: F/F0 image during the application of NE (200 M) and C: Calcium signal of eight astrocytes in response to NE-application.
GaAsP Photomultipliers (Hamamatsu) using a 2-photon excitation microscopy unit (TriMScope 1, LaVision BioTec) connected to an Axioscope FS2 (Zeiss). 2-Photon excitation was achieved with a Ti:Sapphire Laser (SpectraPhysics MaiTai BB) at 850 nm. Fluorescence signals of GCaMP3-expressing astrocytes were detected through a 531/40 nm band pass emission filter (AHF Analysentechnik AG). 2.6. Drugs Electrolytes for aCSF (see above) and tamoxifen were purchased from Sigma–Aldrich (Taufkirchen, Germany) and Merck chemicals (Darmstadt, Germany). Agonists and antagonists were brought from Tocris Bioscience (Bristol, UK) and bath applied at concentrations indicated in the text. 2.7. Data analysis Data are expressed as mean ± SEM. Statistical comparison was performed with the SigmaPlot software (Systat Software, Inc.). Statistical significance (paired t-tests) was assumed if p < 0.05. 3. Results 3.1. Astrocytic norepinephrine induced calcium responses in the ventrolateral medulla In a first set of experiments, we tested whether norepinephrine (NE) has a direct effect on astrocytes in the ventrolateral medulla. Therefore, we applied NE (10 M) in the presence and absence of tetrodotoxin (TTX) that blocks neuronal action potentials. There was no significant change of the amplitude of the NE-induced cal-
cium rise in astrocytes (0.514 ± 0.216 (F/F0 ) in control (CTRL) and 0.521 ± 0.216 (F/F0 ) in TTX; n.s.; n = 6 slices / 3 animals; Fig. 1C). Astrocytes often respond to neuromodulators by a release of calcium from intracellular stores (Härtel et al., 2009). To test if the NE-induced response is mediated by an intracellular calcium release, we applied cyclopiazonic acid (CPA), a blocker of the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) that depletes intracellular calcium stores (Le Pichon and Chesler, 2014). In the presence of CPA, the amplitude of the calcium response in astrocytes was significantly diminished (CTRL 0.453 ± 0.191 (F/F0 ) vs. CPA 0.026 ± 0.0360 (F/F0 ); p = 0.003; n = 7 slices / 4 animals; Fig. 1D) indicating that intracellular calcium release is the major source of the astrocytic NE-induced calcium signal. Different adrenergic receptors have been described in astrocytes of the brainstem including the ␣1-adrenergic receptor that is known to cause release of calcium from intracellular stores. Indeed application of the ␣1-adrenregic receptor blocker prazosin (50 M; (Nilsson et al., 1992)) significantly reduced the NE-induced calcium response (0.283 ± 0.111 (F/F0 ) CTRL vs. 0.092 ± 0.130 (F/F0 ) in prazosin; P = 0.008; n = 6 slices / 3 animals; Fig. 1E) indicating that NE-acts via G protein coupled Phospholipase C-Inositol trisphosphate cascade (Gq-PLC-IP3). Imaging using camera based detection and AM-ester dyes bears the risk of contamination of light from neighboring cells (see (Oku et al., 2015) this issue). To overcome this problem we confirmed NE-induced calcium signals in astrocytes that express the genetically encoded calcium sensor GCaMP3 controlled by the glutamate/aspartate transporter locus (Glast, EAAT1); n = 6 slices / 6 animals. After application of NE (50– 200 M) astrocytes responded with a typical oscillatory calcium signal (Fig. 2), that was earlier described for type 1 metabotropic glutamate receptor agonists (Schnell et al., 2011).
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008
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Fig. 3. A: Schematic drawing of astrocytic norepinephrine signaling. B: Table of the expression of key players of the adrenergic signaling in the medulla based on cell type-specific next generation sequencing (Schnell et al., 2015).
4. Discussion Norepinephrine (NE) increases intracellular calcium in the preBötC astrocytes. This astrocytic calcium signal might mediate an indirect contribution to the well-known stimulatory NE effect on the respiratory network activity as suggested for metabotropic glutamate receptors (Schnell et al., 2011). Norepinephrine acts via ␣1-adrenoreceptors that release calcium from intracellular stores via the Gq-PLC-IP3 pathway (Fig. 3). Indeed, ␣1-adrenoreceptors were identified on Bergmann-Glia (Kirischuk et al., 1996) or astrocytes in the hippocampus (Duffy and MacVicar, 1995). These receptors were also suggested to be responsible for the calcium rise in the preBötC (Ruangkittisakul et al., 2009). Our data supports this concept and strengthen the hypothesis by showing that the calcium signal induced by NE does not result from an optical signal contamination from neighboring neuronal processes (Fig. 2). Moreover, the persistence of the NE-induced calcium signal in the presence of TTX excludes the possibility that the calcium rise in the astrocyte is mediated indirectly via activation of NE-receptors expressing neurons that release e.g., glutamate. Next generation sequencing from brainstem astroglial mRNA (Schnell et al., 2015) showed additional astrocytic expression of ␣2- and 1-adrenoreceptors (Fig. 3). ␣2- and 1-adrenoreceptors, which antagonistically regulate cAMP levels via Gi/o and GS , respectively, might have contributed to the remaining calcium signal after blockade of ␣1-adrenoreceptors by prazosin (Fig. 1E). Although astrocytic calcium levels potentially can be modulated by the exchange protein directly activated by
cAMP (EPAC) pathway (Di Cesare et al., 2006), the current data do not suggest a major contribution of the EPAC signaling pathway. 4.1. Technical consideration To induced GCaMP3 expression in astrocytes, tamoxifen was administered to lactating mice in order to induce GCaMP3 expression in neonatal mice (Leone et al., 2003; Hirrlinger et al., 2006). The tamoxifen-inducible Cre/loxP gene recombination system is frequently used to allow temporal control of gene expression (Jahn et al., 2015) and increases the cell-type specificity of the expression of genetically encoded calcium sensors in astrocytes (Paukert et al., 2014). However, there are reports of unspecific side effects of tamoxifen that have been reported e.g., in cell culture. With respect to astroglial calcium signaling, tamoxifen can influence and interfere with glutamate transporters in astrocytes (Sato et al., 2008; Karki et al., 2013), via upregulation of expression (Karki et al., 2013) or via decrease of effective glutamate transport (Sato et al., 2008). Indeed it has to be assumed that during the induction phase, brain tissue levels of tamoxifen reach concentrations comparable with these culture experiments (Iusuf et al., 2011). More important for our NE-experiments, tamoxifen like other estrogen receptor agonists, can further interfere with the astroglial intracellular calcium signaling cascade by inhibition of the PKC (O’Brian et al., 1985). Thus, we cannot exclude that glutamate signaling was temporarily altered. However, it has been shown in other studies that tamoxifen did not mediate any persistent effect on glutamate transport
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008
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(measures by means of glutamate receptor currents) that could not be explained by the induction of recombination (Saab et al., 2012), making an acute effect that impairs our interpretation of the experiments unlikely. Indeed, we did not observe an increase of spontaneous calcium oscillation in astrocyte in mice with GCaMP3 expression in comparison to the current experiments with Oregon green calcium indicator nor to our earlier data (Härtel et al., 2009; Schnell et al., 2011), suggesting that glutamate levels in the slice preparation of GCaMP3-expressing mice are normal. However, in the current paper GCaMP3 expressing astrocytes (Paukert et al., 2014) were used to confirm that NE-induced calcium signals results indeed from astrocytes and not from the neighboring neuropil (Oku et al., 2015). This aim could be achieved, and we are confident not to have overlooked a relevant factor. 4.2. Implications for respiratory physiology and pathophysiology Since norepinephrine has recently been shown to be a major factor controlling the responsiveness of astrocytes to neuronal network activity (Paukert et al., 2014) we suggest that, when interpreting the role of norepinephrine in healthy and disease states of the respiratory network, astroglial calcium signaling should be taken into consideration. This might be particularly relevant in mouse models of the RETT syndrome, in which the respiratory phenotype depends on Mecp2 levels in astrocytes (Lioy et al., 2011). Thus, the significant changes of norepinephrine concentration that occurs in Mecp2-deficient mice (Viemari et al., 2005) can be expected to alter the network activity both directly and indirectly by changing astroglial calcium signaling and potentially also the levels of glio-transmitters like ATP or glutamate (Volterra et al., 2014). Acknowledgements We thank A.-A. Grützner for technical support. The authors are grateful to D. Rhode and T. Vogelgesang for animal husbandry and tamoxifen treatments (University of Saarland). The work was supported by the “Deutsche Forschungsgemeinschaft” through DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) and personal grant to S.H. (Hu797/5-1). The authors are grateful to M. Götz (Munich) and D. Bergles (Baltimore) for providing mice that founded the breeding of the transgenic mice used for our experiments. References Al-Zubaidy, Z.A., Erickson, R.L., Greer, J.J., 1996. Serotonergic and noradrenergic effects on respiratory neural discharge in the medullary slice preparation of neonatal rats. Pflugers Arch. 431, 942–949. Bianchi, A.L., Denavit-Saubie, M., Champagnat, J., 1995. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75, 1–45. Dallwig, R., Deitmer, J.W., 2002. Cell-type specific calcium responses in acute rat hippocampal slices. J. Neurosci. Methods 116, 77–87. Dallwig, R., Vitten, H., Deitmer, J.W., 2000. A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28, 247–259. Di Cesare, A., Del Piccolo, P., Zacchetti, D., Grohovaz, F., 2006. EP2 receptor stimulation promotes calcium responses in astrocytes via activation of the adenylyl cyclase pathway. Cell. Mol. Life Sci. CMLS 63, 2546–2553. Duffy, S., MacVicar, B.A., 1995. Adrenergic calcium signaling in astrocyte networks within the hippocampal slice. J. Neurosci. 15, 5535–5550. Errchidi, S., Monteau, R., Hilaire, G., 1991. Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study. J.Physiol. 443, 477–498. Funk, G.D., Huxtable, A.G., Lorier, A.R., 2008. ATP in central respiratory control: a three-part signaling system. Respir. Physiol. Neurobiol. 164, 131–142. Funk, G.D., Zwicker, J.D., Selvaratnam, R., Robinson, D.M., 2011. Noradrenergic modulation of hypoglossal motoneuron excitability: developmental and putative state-dependent mechanisms. Arch. Ital. Biol. 149, 426–453. Gomeza, J., Hülsmann, S., Ohno, K., Eulenburg, V., Szoke, K., Richter, D., Betz, H., 2003. Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition. Neuron 40, 785–796.
5
Grass, D., Pawlowski, P.G., Hirrlinger, J., Papadopoulos, N., Richter, D.W., Kirchhoff, F., Hülsmann, S., 2004. Diversity of functional astroglial properties in the respiratory network. J. Neurosci. 24, 1358–1365. Härtel, K., Schnell, C., Hülsmann, S., 2009. Astrocytic calcium signals induced by neuromodulators via functional metabotropic receptors in the ventral respiratory group of neonatal mice. Glia 57, 815–827. Härtel, K., Singaravelu, K., Kaiser, M., Neusch, C., Hülsmann, S., Deitmer, J.W., 2007. Calcium influx mediated by the inwardly rectifying K+ channel Kir4.1 (KCNJ10) at low external K+ concentration. Cell Calcium 42, 271–280. Hilaire, G., Viemari, J.C., Coulon, P., Simonneau, M., Bevengut, M., 2004. Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents. Respir. Physiol. Neurobiol. 143, 187–197. Hirrlinger, P.G., Scheller, A., Braun, C., Hirrlinger, J., Kirchhoff, F., 2006. Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2. Glia 54, 11–20. Hirrlinger, P.G., Scheller, A., Braun, C., Quintela-Schneider, M., Fuss, B., Hirrlinger, J., Kirchhoff, F., 2005. Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice. Mol. Cell Neurosci. 30, 291–303. Hülsmann, S., Oku, Y., Zhang, W., Richter, D.W., 2000. Metabolic coupling between glia and neurons is necessary for maintaining respiratory activity in transverse medullary slices of neonatal mouse. Eur. J. Neurosci. 12, 856–862. Huxtable, A.G., Zwicker, J.D., Alvares, T.S., Ruangkittisakul, A., Fang, X., Hahn, L.B., Posse de Chaves, E., Baker, G.B., Ballanyi, K., Funk, G.D., 2010. Glia contribute to the purinergic modulation of inspiratory rhythm-generating networks. J. Neurosci. 30, 3947–3958. Iusuf, D., Teunissen, S.F., Wagenaar, E., Rosing, H., Beijnen, J.H., Schinkel, A.H., 2011. P-glycoprotein (ABCB1) transports the primary active tamoxifen metabolites endoxifen and 4-hydroxytamoxifen and restricts their brain penetration. J. Pharmacol. Exp. Ther. 337, 710–717. Jahn, H.M., Scheller, A., Kirchhoff, F., 2015. Genetic control of astrocyte function in neural circuits. Front. Cell. Neurosci. 9. Karki, P., Webb, A., Smith, K., Lee, K., Son, D.-S., Aschner, M., Lee, E., 2013. cAMP response element-binding protein (CREB) and nuclear factor B mediate the tamoxifen-induced up-regulation of glutamate transporter 1 (GLT-1) in rat astrocytes. J. Biol. Chem. 288, 28975–28986. Kirischuk, S., Tuschick, S., Verkhratsky, A., Kettenmann, H., 1996. Calcium signalling in mouse Bergmann glial cells mediated by alpha1-adrenoreceptors and H1 histamine receptors. Eur. J. Neurosci. 8, 1198–1208. Le Pichon, C.E., Chesler, A.T., 2014. The functional and anatomical dissection of somatosensory subpopulations using mouse genetics. Front. Neuroanat., 8. Leone, D.P., Genoud, St., Aanasoski, S., Grausenburger, R., Berger, P., Metzger, D., Macklin, W.B., Chambon, P., Suter, U., 2003. Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells. Mol. Cell. Neurosci. 22, 430–440. Lioy, D.T., Garg, S.K., Monaghan, C.E., Raber, J., Foust, K.D., Kaspar, B.K., Hirrlinger, P.G., Kirchhoff, F., Bissonnette, J.M., Ballas, N., Mandel, G., 2011. A role for glia in the progression of Rett/’s syndrome. Nature advance online publication. Mori, T., Tanaka, K., Buffo, A., Wurst, W., Kuhn, R., Götz, M., 2006. Inducible gene deletion in astroglia and radial glia–a valuable tool for functional and lineage analysis. Glia 54, 21–34. Nilsson, M., Hansson, E., Ronnback, L., 1992. Agonist-evoked Ca2+ transients in primary astroglial cultures—modulatory effects of valproic acid. Glia 5, 201–209. O’Brian, C.A., Liskamp, R.M., Solomon, D.H., Weinstein, I.B., 1985. Inhibition of Protein Kinase C by Tamoxifen. Cancer Res. 45, 2462–2465. Oku Y, Fresemann J, Miwakeichi F, Hülsmann S. (2015) Respiratory calcium fluctuations in low-frequency oscillating astrocytes in the pre-Botzinger complex. Respir Physiol Neurobiol. Paukert, M., Agarwal, A., Cha, J., Doze, V.A., Kang, J.U., Bergles, D.E., 2014. Norepinephrine controls astroglial responsiveness to local circuit activity. Neuron 82, 1263–1270. Robertson, S.D., Plummer, N.W., de Marchena, J., Jensen, P., 2013. Developmental origins of central norepinephrine neuron diversity. Nat. Neurosci. 16, 1016–1023. Ruangkittisakul, A., Okada, Y., Oku, Y., Koshiya, N., Ballanyi, K., 2009. Fluorescence imaging of active respiratory networks. Respir. Physiol. Neurobiol. 168, 26–38. ˇ Saab, A.S., Neumeyer, A., Jahn, H.M., Cupido, A., Simek, A.A.M., Boele, H.-J., Scheller, A., Le Meur, K., Götz, M., Monyer, H., Sprengel, R., Rubio, M.E., Deitmer, J.W., De Zeeuw, C.I., Kirchhoff, F., 2012. Bergmann Glial AMPA receptors are required for fine motor coordination. Science 337, 749–753. Sato, K., Saito, Y., Oka, J.-, i, O., hwada, T., Nakazawa, K., 2008. Effects of Tamoxifen on L-Glutamate Transporters of Astrocytes. J. Pharmacol. Sci. 107, 226–230. Schnell, C., Fresemann, J., Hülsmann, S., 2011. Determinants of functional coupling between astrocytes and respiratory neurons in the pre-Botzinger complex. PLoS One 6, e26309. Schnell, C., Shahmoradi, A., Wichert, S.P., Mayerl, S., Hagos, Y., Heuer, H., Rossner, M.J., Hülsmann, S., 2015. The multispecific thyroid hormone transporter OATP1C1 mediates cell-specific sulforhodamine 101-labeling of hippocampal astrocytes. Brain Struct. Funct. 220, 193–203. Szöke, K., Härtel, K., Grass, D., Hirrlinger, P.G., Hirrlinger, J., Hülsmann, S., 2006. Glycine transporter 1 expression in the ventral respiratory group is restricted to protoplasmic astrocytes. Brain Res. 1119, 182–189. Viemari, J.C., Ramirez, J.M., 2006. Norepinephrine differentially modulates different types of respiratory pacemaker and nonpacemaker neurons. J. Neurophysiol. 95, 2070–2082.
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Viemari, J.C., Roux, J.C., Tryba, A.K., Saywell, V., Burnet, H., Pena, F., Zanella, S., Bevengut, M., Barthelemy-Requin, M., Herzing, L.B., Moncla, A., Mancini, J., Ramirez, J.M., Villard, L., Hilaire, G., 2005. Mecp2 deficiency disrupts norepinephrine and respiratory systems in mice. J. Neurosci. 25, 11521–11530. Volterra, A., Liaudet, N., Savtchouk, I., 2014. Astrocyte Ca2+ signalling: an unexpected complexity. Nat. Rev. Neurosci. 15, 327–335.
Winter, S.M., Fresemann, J., Schnell, C., Oku, Y., Hirrlinger, J., Hülsmann, S., 2009. Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices. Pflugers Arch. 458, 459–469. Zhao, M.G., Hülsmann, S., Winter, S.M., Dutschmann, M., Richter, D.W., 2006. Calcium-regulated potassium currents secure respiratory rhythm generation after loss of glycinergic inhibition. Eur. J. Neurosci. 24, 145–154.
Please cite this article in press as: Schnell, C., et al., Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.10.008