Serotonin Receptor 4 Regulates Hippocampal Astrocyte Morphology and Function
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Received: 18 November 2019 Revised: 15 October 2020 Accepted: 19 October 2020 DOI: 10.1002/glia.23933 RESEARCH ARTICLE Serotonin receptor 4 regulates hippocampal astrocyte morphology and function Franziska E. Müller1 | Sophie K. Schade1 | Volodymyr Cherkas1 | Laura Stopper2 | Björn Breithausen3 | Daniel Minge3 | Hristo Varbanov4 | Christian Wahl-Schott4 | Svitlana Antoniuk1 | Catia Domingos3 | Valérie Compan5 | Frank Kirchhoff2 | Christian Henneberger3,6,7 | Evgeni Ponimaskin1,8# | Andre Zeug1 1Cellular Neurophysiology, Hannover Medical School, Hannover, Germany Abstract 2Department of Molecular Physiology, Center Astrocytes are an important component of the multipartite synapse and crucial for for Integrative Physiology and Molecular proper neuronal network function. Although small GTPases of the Rho family are Medicine (CIPMM), University of Saarland, Homburg, Germany powerful regulators of cellular morphology, the signaling modules of Rho-mediated 3Institute of Cellular Neurosciences, Medical pathways in astrocytes remain enigmatic. Here we demonstrated that the serotonin School, University of Bonn, Bonn, Germany receptor 4 (5-HT4R) is expressed in hippocampal astrocytes, both in vitro and in vivo. 4Institute of Neurophysiology, Hannover Medical School, Hannover, Germany Through fluorescence microscopy, we established that 5-HT4R activation triggered 5 Department of Sciences, Brain, Anorexia & RhoA activity via Gα13-mediated signaling, which boosted filamentous actin assem- Addiction, Nîmes University, Nîmes, France bly, leading to morphological changes in hippocampal astrocytes. We investigated 6German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany the effects of these 5-HT4R-mediated changes in mixed cultures and in acute slices, 7 Institute of Neurology, University College in which 5-HT4R was expressed exclusively in astrocytes. In both systems, 5-HT4R- London, London, UK RhoA signaling changed glutamatergic synaptic transmission: It increased the fre- 8Institute of Neuroscience, Lobachevsky State University of Nizhny Novgorod, Nizhny quency of miniature excitatory postsynaptic currents (mEPSCs) in mixed cultures and Novgorod, Russia reduced the paired-pulse-ratio (PPR) of field excitatory postsynaptic potentials Correspondence (fEPSPs) in acute slices. Overall, our present findings demonstrate that astrocytic Evgeni Ponimaskin, Cellular Neurophysiology, 5-HT4R-Gα13-RhoA signaling is a previously unrecognized molecular pathway Hannover Medical School, Hannover, Germany. involved in the functional regulation of excitatory synaptic circuits. Email: [email protected] KEYWORDS Funding information actin, astrocytes, neuronal excitability, RhoA, serotonin Deutsche Forschungsgemeinschaft, Grant/ Award Numbers: FOR2795, HE6949/1; HE6949/3, PO732, SFB1089 B03, SFB870 B05, SPP1757, ZE994/2 1 | INTRODUCTION Astrocytes are an important component of the brain circuitry. They # Evgeni Ponimaskin and Andre Zeug are equally contributing corresponding authors. are highly heterogeneous, exhibiting variations in their appearance This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. GLIA published by Wiley Periodicals LLC Glia. 2020;1–18. wileyonlinelibrary.com/journal/glia 1 2 MÜLLER ET AL. and functionality across brain regions and even within substructures, In the present study, by combining quantitative molecular micros- such as the hippocampus (Amundson, Goderie, & Kimelberg, 1992; copy, time-lapse Förster resonance energy transfer (FRET) imaging, Lin et al., 2017; Wallraff, Odermatt, Willecke, & Steinhäuser, 2004). and biochemical approaches, we demonstrated that 5-HT4R was Astrocytes contribute to a multitude of brain processes by modulating expressed in astrocytes of the mouse hippocampus, and that 5-HT4R- synaptic plasticity and neuronal activity (Alfonso Araque et al., 2014; Gα13 signaling in astrocytes increased RhoA activity leading to accu- Rusakov, Bard, Stewart, & Henneberger, 2014; Ullian, Sapperstein, mulation of filamentous actin structures. We also identified the role Christopherson, & Barres, 2001), controlling extra-synaptic space of 5-HT4R-Gα13-RhoA signaling in the regulation of astrocytic mor- (Flores-Méndez, Mendez-Flores, & Ortega, 2016) and neurotransmit- phology. Moreover, electrophysiological experiments in mixed cul- ter clearance (Rose et al., 2018; Sibille, Pannasch, & Rouach, 2014), tures and in acute slices revealed that astrocytic 5-HT4R modifies the regulating cerebral blood flow (Mishra, 2017), ensuring energy supply function of excitatory hippocampal synapses. to neurons, and orchestrating the rhythms of neuronal firing patterns (Camandola, 2018; Lee et al., 2014; Sheikhbahaei et al., 2018; Stobart & Anderson, 2013). 2 | MATERIALS AND METHODS Over a century ago, astrocytes were first classified as fibrous or protoplasmic based on their morphology (Andriezen, 1893). In 2.1 | Animals recent years, novel approaches and technological progress have enabled great advance in our understanding of the complexity of For all experiments wild type and 5-HT4R-ko animals from the strain astrocyte morphology and function (Wu, Pan, Zuo, Li, & B6-Htr4tm1comp (Compan et al., 2004) on a C57BL/6J background Hong, 2017; Zhang & Barres, 2010). Astrocytes ensheath a vast were used. Animals were housed and cared for in accordance to direc- number of synapses, through whichtheyactivelycontrolsynaptic tive 2010/63/EU. Mice were kept in a 14 hr light and 10 hr dark cycle plasticity and transmission (Araque, Parpura, Sanzgiri, & with lights on starting at 7 a.m. Animals had access to food and water Haydon, 1999; Chung, Allen, & Eroglu, 2015; Halassa, Fellin, ad libitum and were kept under standard conditions at 22 ± 2C room Takano, Dong, & Haydon, 2007). Astrocyte morphology is regulated temperature (RT) with 55 ± 5% humidity. Stereotactic injections were by small GTPases of the Rho family—including Cdc42, Rac1, and done under allowance given by the Lower Saxony State Office for RhoA (for review see Zeug et al., 2018). Experimental findings Consumer Protection and Food Safety (TVA16/2206). All experiments suggest that Rac1 and Cdc42 promote filopodia formation and were conducted according to the relevant guidelines. For cell culture outgrowth, whereas RhoA activation triggers filopodia retraction male and female pups were used while in all other experiments only (Hall, 1998; Hall, 2005; Mackay & Hall, 1998). male mice were investigated. The importance of Rho GTPases in morphogenesis is widely accepted; however, the upstream signaling components of Rho- mediated pathways in astrocytes remain enigmatic. We previously 2.2 | Cell culture demonstrated that serotonin receptor 4 (5-HT4R) is coupled to the heterotrimeric Gα13 protein, leading to selective activation of the Primary astrocyte cell cultures were prepared as previously described small GTPase RhoA, and promoting morphological changes in by Wu et al. (Wu et al., 2014) with slight modifications. Whole brains neuroblastoma cells and hippocampal neurons (Kvachnina, 2005; were taken from mice between P1-3 and cells from dissociated hippo- Ponimaskin, Profirovic, Vaiskunaite, Richter, & Voyno-Yasenetskaya,- campi were seeded at a density of 5 × 104 cells per 12 mm glass cov- 2002). In addition to Gα13, 5-HT4R can activate heterotrimeric GαS erslip for microscopy in 500 μl plating medium (49 ml Minimal proteins to induce cAMP-PKA signaling (Bockaert, Claeysen, Bécamel, Essential Medium, 1 ml B-27, 500 μl 200 mM glutamine, 500 μl Dumuis, & Marin, 2006; Muller & Jacobs, 2009). Moreover, 5-HT4R 100 mM sodium pyruvate, 5 μ/ml penicillin, 5 mg/ml streptomycin). signaling can occur independently of G proteins, leading to activation On DIV3 the entire plating medium was replaced with 1 ml mainte- of proto-oncogene tyrosine kinase Src and extracellular signal- nance medium (49 ml Neurobasal-A medium, 1 ml B-27, 500 μl regulated kinase (ERK), and subsequent pERK1/2 phosphorylation 200 mM glutamine, 5 μ/ml penicillin, 5 mg/ml streptomycin). With (Barthet et al., 2007). exception of the shRNA experiments, 1/2 of the medium was Prior reports describe 5-HT4R expression by cultured astrocytes exchanged on DIV11 with maintenance medium prior to infection of in vitro (Parga, Rodriguez-Pallares, Muñoz, Guerra, & Labandeira- the cells. Astrocytes were used for experiments on DIV14-17. Mixed Garcia, 2007). Moreover, microarray analysis confirms 5-HT4R mRNA hippocampal cultures were obtained from dissociated hippocampi of expression in astrocytes in the brain at postnatal day (P) 7 and P17, neonatal mice at P0-1 using an optimized protocol (Kobe et al., 2012). and in cultured astrocytes at days in vitro (DIV) 12 (Cahoy At DIV7 cells were infected with AAVs. Cell cultures were maintained et al., 2008). A more recent study revealed age-independent 5-HT4R at 37 C in a humidified incubator in a 5% CO2 atmosphere until they expression in astrocytes within several regions of the mouse brain were used for experiments at DIV12. During microscopy, cells were (Boisvert, Erikson, Shokhirev, & Allen, 2018). However, the impact of kept in a balanced salt solution containing 115 mM NaCl, 5.4 mM KCl, 5-HT4R activity on astrocytic morphology and function remains 1 mM MgCl2, 2 mM CaCl2, and 20 mM HEPES, adjusted to pH 7.4 largely unexplored. and 290 mOsm with glucose. MÜLLER ET AL. 3 2.3 | Immunohistochemistry and 2.4