Aquaporin-4 Regulates Extracellular Space Volume Dynamics During High-Frequency Synaptic Stimulation: A Gene Deletion Study in Mouse Hippocampus NADIA NABIL HAJ-YASEIN, 1,2 VIDAR JENSEN, 1,2 IVAR ØSTBY, 3 STIG W. OMHOLT, 4 JUHA VOIPIO, 5 KAI KAILA, 5,6 OLE P. OTTERSEN, 2 ØIVIND HVALBY, 7 AND ERLEND A. NAGELHUS 1,2,8 * 1 Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, Oslo, Norway 2 Centre for Molecular Biology and Neuroscience, Letten Centre, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway 3 Department of Mathematical Sciences and Technology, Norwegian University of Life Sciences, ˚ As, Norway 4 Centre for Integrative Genetics (CIGENE), Norwegian University of Life Sciences, ˚ As, Norway 5 Department of Biosciences, University of Helsinki, Helsinki, Finland 6 Neuroscience Center, University of Helsinki, Helsinki, Finland 7 Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway 8 Department of Neurology, Oslo University Hospital, Oslo, Norway KEY WORDS activity; AQP4; astrocytes; brain; glia; shrinkage; swelling; water transport ABSTRACT Little is known about the physiological roles of aquaporin-4 (AQP4) in the central nervous system. AQP4 water chan- nels are concentrated in endfeet membranes of astrocytes but also localize to the fine astrocytic processes that abut central synapses. Based on its pattern of expression, we predicted that AQP4 could be involved in controlling water fluxes and changes in extracellular space (ECS) volume that are associated with activation of excitatory pathways. Here, we show that deletion of Aqp4 accentuated the shrinkage of the ECS that occurred in the mouse hippocam- pal CA1 region during activation of Schaffer collateral/com- missural fibers. This effect was found in the stratum radia- tum (where perisynaptic astrocytic processes abound) but not in the pyramidal cell layer (where astrocytic processes constitute but a minor volume fraction). For both genotypes the ECS shrinkage was most pronounced in the pyramidal cell layer. Our data attribute a physiological role to AQP4 and indicate that this water channel regulates extracellular volume dynamics in the mammalian brain. V V C 2012 Wiley Periodicals, Inc. INTRODUCTION What are the factors that regulate the extracellular space (ECS) volume in brain? Despite its importance, this question has not received much attention. One as- pect is the baseline ECS volume; another fundamental aspect is the ECS volume dynamics coupled to synaptic activity. More than three decades ago it was shown that evoked synaptic activity is followed by a rapid shrinkage of the ECS (Dietzel et al., 1980). The shrinkage is sub- stantial, amounting to 4–30%, depending on the mode of stimulation, type of preparation, and area under investi- gation (Dietzel et al., 1980; Holthoff and Witte, 1996; Sykova et al., 2003; Viitanen et al., 2010). ECS volume changes have a significant impact on the concentration of extracellular solutes and vice versa. Syn- aptic stimulation causes release of a number of solutes that are removed through cotransport with water (Mac- Aulay and Zeuthen, 2010). Essential among these is K 1 , which is cleared by a multitude of mechanisms including chloride coupled uptake into glia (Kimelberg, 2010; Kofuji and Newman, 2004; Ostby et al., 2009). Likewise, glial uptake of bicarbonate and glutamate is associated with water transport and may contribute to the observed shrinkage of the ECS (Nagelhus et al., 2004). ECS volume changes are also implicated in a number of pathophysio- logical conditions such as spreading depression and are known to precede seizure activity (Olsson et al., 2006). With the discovery of water channels in brain, the question arose whether these are involved in ECS vol- ume dynamics. AQP4—the predominant brain water channel—is enriched in those astrocytic plasma mem- brane domains that face vessels and pia but is also expressed in the delicate astrocyte processes around excitatory synapses (Frigeri et al., 1995; Nielsen et al., 1997). AQP4 water channels in the latter processes are unlikely to be passive bystanders to the local volume dy- namics. Indeed, a study measuring light scattering sug- gested a role for AQP4 in activity-induced volume changes in acute cortical slices (Kitaura et al., 2009). If involved, the perisynaptic AQP4 pool could either curb or accentuate the activity-dependent shrinkage of the ECS. Here, we show that the activity-dependent ECS shrinkage is more pronounced in animals lacking AQP4 The first two authors contributed equally to this work. Grant sponsors: Research Council of Norway, Letten Foundation, Marie Curie Mobility Actions of the European Union, and the Nordic Centre of Excellence Pro- gramme on Molecular Medicine. *Correspondence to: Erlend A. Nagelhus, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, P.O. Box 1137 Blindern, Oslo N- 0318, Norway. E-mail: e.a.nagelhus@ncmm.uio.no Received 29 November 2011; Accepted 14 February 2012 DOI 10.1002/glia.22319 Published online 14 March 2012 in Wiley Online Library (wileyonlinelibrary. com). GLIA 60:867–874 (2012) V V C 2012 Wiley Periodicals, Inc.