Despite increased recognition of the importance and heterogeneity of astrocyte functions throughout the central nervous system (CNS) relatively little attention has been paid to morphological diversity among astrocytes. Recent studies have indicated that subsets of astrocytes are involved in glial-axonal interactions critical to both development and reinnervation of the rat olfactory bulb. Here, we have characterized the morphologies and distribution of astrocytes within anatomically and functionally distinct layers of the adult main olfactory bulb (MOB). Using a known immunohistochemical marker for astrocytes, glial fibrillary acidic protein (GFAP), and the classic gold sublimate method, we identified six astrocyte subtypes based on their morphology and distribution: (1) unipolar, (2) irregular, (3) wedge-shape, (4) circular, (5) semicircular, and (6) elongate. Unipolar, irregular and wedge-shape astrocytes have not been previously described in the CNS. The unipolar and irregular types are located exclusively in the olfactory nerve layer. Wedge-shape astrocytes are unique to, and are the major subtype in, the glomerular layer. These three morphologically unique astrocyte subtypes may correspond to olfactory nerve layer (ONL) and glomerular layer (GL) astrocytes, which express molecules that regulate axonal growth or synaptogenesis during development and/or regeneration of the olfactory nerve. In the glomerular layer, astrocytes are highly organized with respect to the glomeruli. Individual astrocytes are loyal to a single glomerulus. In the external plexiform layer, astrocytes are spaced relatively uniformly. In the granule cell layer, astrocytes appear to compartmentalize granule cell aggregates, recently shown to be coupled by tight junctions. The distribution and patterns of astrocyte processes and the density of GFAP immunoreactivity are distinctive for each of the layers of the olfactory bulb. The spacing of astrocytes and the organization of their processes may be important to compartmentalization of neuronal functions. High levels of GFAP immunoreactivity correlated with layers of high neuronal plasticity. The morphological diversity and differential distribution of astrocytes in the olfactory bulb reported here support growing evidence for functional diversity of astrocytes and important interactions among specific astrocyte and neuron subtypes. It is reasonable to hypothesize, therefore, that as for neurons, morphologically distinctive astrocyte subtypes may correspond to functionally specific classes.

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Nerve growth factor (NGF), a classic neurotrophic factor, promotes neuronal survival, maintenance, regeneration and differentiation in the peripheral nervous system and parts of the central nervous system. NGF activity is mediated by cell surface bound receptors including the low affinity NGF receptor (LNGFr) which is expressed by some peripheral and central neurons and is present on peripheral nerve Schwann cells during development and regeneration. The olfactory system is a useful model for the study of the role of LNGFr in neuronal development and regeneration. The growth of olfactory axons into the brain begins in the embryo and continues through the first few postnatal weeks. In mature animals there is persistent turnover and generation of olfactory receptor neurons (ORNs) and continuous growth of new axons into the olfactory bulb. These new axons grow along the preexisting olfactory pathway. In the mature olfactory system, LNGFr has been observed in the glomerular layer of the olfactory bulb, the target of ORNs. However, neither the cellular localization nor the developmental expression of LNGFr has been characterized. Here, we tested the hypothesis that LNGFr expression is developmentally regulated in the olfactory nerve and is reinduced following injury to the mature olfactory nerve. LNGFr-immunoreactivity (IR) was first observed in the olfactory mucosa at embryonic day (E)13 and in the olfactory nerve at E14. LNGFr-IR increased in the nerve during embryonic development, began to decrease at around postnatal day (P)5 and was scarcely detectable in normal adults. The staining pattern suggests that LNGFr is located on the olfactory nerve Schwann cells. Streaks of LNGFr-IR were present in the adult olfactory nerve. We reasoned that these streaks might represent transient reexpression of LNGFr associated with normal olfactory neuron turnover and replacement. Consistent with this hypothesis, LNGFr was robustly reexpressed in the adult olfactory nerve following lesion of the olfactory epithelium. Starting late in development (E21) and in the adult, LNGFr-IR was also observed on fibers in deep layers of the olfactory bulb. LNGFr-IR was also observed in neurons of the nucleus of the diagonal band (NDB) in the basal forebrain. NDB is the sole source of cholinergic afferents of the olfactory bulb. Thus, we tested the hypothesis that LNGFr in the deep layers of the olfactory bulb is located on NDB axons by making lesions of NDB. Following the lesion, LNGFr-IR disappeared in the deep layers of the olfactory bulb but remained in the glomerular layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Olfactory bulb (OB) glomeruli have long been considered functional units in the processing of odor information. Recently, it has been shown that axons from olfactory receptor neurons (ORNs) expressing the same odorant receptor gene converge onto two or a few topographically fixed glomeruli in the OB. The interactions between ORN axons, mitral/tufted cell dendrites, juxtaglomerular (JG) cells, and glial cells during the development of glomeruli is of great importance in light of this receptor gene glomerular topography in the primary olfactory projection. To explore the development of mammalian olfactory glomeruli, we investigated the relationships among radial glia (RG), astrocytes, ORNs, JG cells, mitral/tufted cell dendrites, and olfactory Schwann cells throughout embryonic and early postnatal development. Our results indicate that glomeruli are formed through an invariant sequence of cellular events: (1) pioneering ORN axons contact the rostral telencephalon at approximately E11-14, which coincides with the onset of morphologic changes in telencephalic RG; (2) at E15-16, RG branch and begin to form two plexuses, one located in the subventricular layer and the other superficial to the presumptive mitral cell layer; (3) at E17-18, ORN axons accumulate in a dense band superficial to the outer radial glia plexus; (4) at E19-20, processes from RG and astrocytes begin to ramify to form glial tufts, or glial glomeruli. Coincident with the formation of these glial glomeruli, ORN axons intermingle with the glial processes and form proto-glomeruli; (5) at E21 to P0, JG cells begin to migrate into position surrounding glomeruli, (6) and at P4, the apical tuft of mitral cells becomes restricted to a single glomerulus. Interestingly, glomerular development also occurs in a distinct rostral to caudal gradient. That is, glomeruli in the rostral OB develop earlier than those in the caudal OB, but the sequence of cellular events at any point in the bulb is invariant. These results demonstrate that glomeruli are formed in a specific spatiotemporal sequence beginning with ORN axon-glia contacts, then JG cell arrival, and finally mitral cell apical dendrite restriction.

The cellular localizations of the synthetic [3-hydroxyanthranilic acid oxygenase (3HAO)] and degradative [quinolinic acid phosphoribosyltransferase (QPRT)] enzymes of the endogenous excitotoxin quinolinic acid were studied in the adult rat main olfactory bulb by immunohistochemical techniques. 3HAO and QPRT were expressed only in astrocytes. The two enzymes were differentially expressed by astrocytes in a complementary pattern: 3HAO staining was strongest at the glomerular-external plexiform layer junction; QPRT staining was strongest at the glomerular-olfactory nerve layer junction. The complementary distributions of these metabolic enzymes suggests that there could be a gradient of quinolinic acid across the glomerular layer of the main olfactory bulb. Such a gradient could function to restrict the ingrowth of new olfactory axons to the glomeruli and/or to stabilize the formation of new synapses.