Role of Astroglial Extracellular Matrix in the Formation of Rat Olfactory Bulb Glomeruli

Abstract

We describe the development of the olfactory bulb (OB) glomeruli, focusing on the role of purported astroglial-associated boundary molecules. as potential guidance factors in glomeruli formation. Glomeruli are unusual structures within the OB formed by extremely tight bundles of afferent fibers that fail to grow deeply into the brain. Using olfactory marker protein antibodies, we found that olfactory axons of stage E17-E18 rat embryos were organized into long strands at the outermost edge of the bulb. By E20-E21 the axons clustered and formed small spheres and by PD1 they gathered into distinct glomeruli. Prior to and during the transformation of olfactory afferents into glomeruli, there was an enhanced expression of cytotactin/tenascin (CT) and chondroitin sulfate-containing proteoglycan (CSPG) associated with VIM/GFAP-positive astroglial cell processes at the base of the glomeruli. During later stages of development (PD3-PD9), the GFAP-positive astroglial processes beneath and within the base of the glomeruli showed intensified GFAP staining while maintaining expression of CT and CS-PG. Some astroglial processes had extended and others went even further peripherally into the olfactory nerve layer (ONL) to encapsulate and fully infiltrate the glomeruli. By PD9, CS-PG and CT levels increased inside and outside the glomeruli. However, by PD15 both molecules had decreased inside the glomeruli but remained highly concentrated around the glomeruli to form rings. Thus, at later stages, axon/glia interactions which result in astroglial rearrangement and matrix alterations in relation to the glomeruli occur. Western blots of stage E20 bulb proteins revealed the presence of three proteoglycan core proteins each containing chondroitin-6-sulfate moieties. In addition, a type of keratan sulfate (KS) glycosaminoglycan was present in a core protein not associated with chondroitin-6-sulfate. Our data suggest that astroglial-associated ECM molecules CT and CSPG, and perhaps KS-PG, present in the core of the early developing OB, could form a molecular "wall" that helps confine ingrowing olfactory axons within the ONL at the outer edge of the astroglial territory. We suggest that the astroglia do not precisely regulate the specific shape of the glomeruli, but rather play a fundamental role in directing the gross position at which formation of the synaptic glomeruli will occur.