Int. J. Devl Neuroscience 22 (2004) 73–86 Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development Eric A. Bushong a,c , Maryann E. Martone b,c , Mark H. Ellisman b,c,∗ a Biomedical Sciences Program, University of California, San Diego, 9500 Gilman Dr., Basic Science Building Rm. 1000, La Jolla, CA 92093-0608, USA b Department of Neurosciences, University of California, San Diego, 9500 Gilman Dr., Basic Science Building Rm. 1000, La Jolla, CA 92093-0608, USA c National Center for Microscopy and Imaging Research, University of California, San Diego, 9500 Gilman Dr., Basic Science Building Rm. 1000, La Jolla, CA 92093-0608, USA Received 20 October 2003; received in revised form 12 December 2003; accepted 15 December 2003 Abstract Mature protoplasmic astrocytes exhibit an extremely dense ramification of fine processes, yielding a ‘spongiform’ morphology. This complex morphology enables protoplasmic astrocytes to maintain intimate relationships with many elements of the brain parenchyma, most notably synapses. Recently, it has been demonstrated that astrocytes establish individual cellular-level domains within the neuropil, with limited overlap occurring between the extents of neighboring astrocytes. The highly ramified nature of protoplasmic astrocytes is closely associated with their ability to create such domains. This study was an attempt to characterize the development of spongiform processes and the establishment of astrocyte domains. A combination of immunolabeling for the astrocyte-specific markers glial fibrillary acidic protein and S100 with intracellular dye labeling in fixed tissue slices allowed for the identification of immature astrocytes and the elucidation of their complete, well-preserved morphologies. We find that during the first two postnatal weeks astrocytes extend stringy, filopodial processes. Fine, spongiform processes appear during the third week. Protoplasmic astrocytes are quite heterogeneous in morphology at 1-week postnatum, but there is a remarkable consistency in morphology by 2 weeks of age. Finally, protoplasmic astrocytes initially extend long, overlapping processes during the first two postnatal weeks. The subsequent elaboration of spongiform processes results in the development of boundaries between neighboring astrocyte domains. Stray processes that encroach on neighboring domains are eventually pruned by 1 month of age. These observations suggest that domain formation is largely the consequence of competition between astrocyte processes, similar to the well-studied competitive interactions between certain neuronal dendritic fields. © 2004 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Maturation; Astrocytes; Morphology 1. Introduction Protoplasmic astrocytes, the predominate glial cell type of gray matter, exhibit a notably intricate morphology in the mature brain, often described as bushy or spongiform. The processes of protoplasmic astrocytes are highly ramified, generating innumerable delicate leaflet processes, which are insinuated between and around the various compo- nents of the nervous tissue. These spongiform elaborations are especially noteworthy for their intimate associations with synapses, particularly of the excitatory, spiny variety (Spacek, 1985; Jones and Greenough, 1996; Ventura and Harris, 1999; Lehre and Rusakov, 2002; Murai et al., 2003). However, astrocytes are increasingly seen as fulfilling many roles within the normal brain, such as providing some ∗ Corresponding author. Tel.: +1-858-534-2251; fax: +1-858-534-7497. E-mail address: mellisman@ucsd.edu (M.H. Ellisman). degree of mechanical integrity to the tissue, maintaining the composition of the extracellular milieu, participating in synaptic function and plasticity, providing neurotransmitters and energy substrates to neurons, and regulating blood flow. Accordingly, their elaborate morphology enables protoplas- mic astrocytes to maintain a position near neuronal somata and dendrites, synapses, blood vessels, and the margins of the nervous system. As for the relationship established be- tween neighboring protoplasmic astrocytes, recent reports have indicated that these cells limit the overlap occurring between their processes and thereby establish individual domains within the neuropil, creating a patchwork of rather exclusive astrocyte territories (Bushong et al., 2002; Ogata and Kosaka, 2002). The consequences of this cellular ar- rangement are unknown, but potentially significant (Dienel and Cruz, 2003). The developmental events that yield such a complex ar- rangement of astrocytic processes are not well understood. 0736-5748/$30.00 © 2004 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2003.12.008