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Charles A Greer

Publications and source records attributed to Charles A Greer.

At least 19 recordsLinked to original sources
PubMed2006

Axon behavior in the olfactory nerve reflects the involvement of catenin-cadherin mediated adhesion.

The projection of olfactory sensory neuron (OSN) axons to the olfactory bulb (OB) is a complex but well-regulated process. Although odorant receptor proteins, and other molecules, are implicated in this process, our understanding remains incomplete. We demonstrate that axons remain restricted to the outer olfactory nerve layer (ONLo) until they are proximal to their target glomeruli, where they enter the inner ONL (ONLi), dividing the ONL into extension and sorting zones. Sorting is likely contingent on cell:cell interactions mediated in part by cell adhesion molecules. The cadherins are a large family of adhesion molecules whose function is contingent on their intracellular binding partners, the catenins, which in turn link to the cytoskeleton. We previously demonstrated that the organization of the cytoskeleton changed as olfactory sensory neuron axons moved from the ONLo to the ONLi. To further assess the role of cadherin mediated adhesion in the developing mouse ONL, we localized alpha-, beta-, gamma-, delta-, and p120-catenins as well as neural cadherin (N-cadherin; CDH2) in the OB. alpha- and beta-catenins are found throughout the OB and are uniform throughout the ONL. In contrast, gamma-catenin and CDH2 are expressed predominantly in the ONLo during perinatal development, but are uniform across the ONL beginning at P7 and into adulthood. Finally, p120- and delta-catenins are expressed in nonoverlapping patterns by olfactory axons and OB neuronal dendrites, respectively. We conclude that gamma-catenin-mediated CDH2 adhesion may influence OSN targeting by restricting axons to the ONLo until they reach the appropriate domain of the OB.

Animals↗
PubMed2006

Viral tracing identifies distributed columnar organization in the olfactory bulb.

Olfactory sensory neurons converge onto glomeruli in the olfactory bulb (OB) to form modular information processing units. Similar input modules are organized in translaminar columns for other sensory modalities. It has been less clear in the OB whether the initial modular organization relates to a columnar structure in the deeper layers involved in local circuit processing. To probe synaptic connectivity in the OB, we injected a retrograde-specific strain of the pseudorabies virus into the rat OB and piriform cortex. The viral-staining patterns revealed a striking columnar organization that extended across all layers of the OB from the glomeruli to the deep granule cell layer. We hypothesize that the columns represent an extension of the glomerular unit. Specific patterning was observed, suggesting selective, rather than distance-dependent, center-surround connectivity. The results provide a previously undescribed basis for interpreting the synaptic connections between mitral and granule cells within the context of a columnar organization in the OB and have implications for olfactory coding and network organization.

Animals↗
PubMed2006

Disrupted compartmental organization of axons and dendrites within olfactory glomeruli of mice deficient in the olfactory cell adhesion molecule, OCAM.

There is an overall topographic connectivity in the axonal projections of olfactory sensory neurons from the olfactory epithelium (OE) to the olfactory bulb (OB). The molecular determinants of this overall topographic OE-OB connectivity are not known. For 20 years, the intriguing expression pattern of the olfactory cell adhesion molecule (OCAM) has made it the leading candidate as determinant of overall topographic OE-OB connectivity. Here, we have generated a strain of OCAM knockout mice by gene targeting. There were no obvious alterations in the distribution of olfactory sensory neurons within the OE or in the coalescence of axons into specific glomeruli. However, the compartmental organization of dendrites and axons within the glomeruli was disrupted. Surprisingly, the mutant mice exhibited an increase in olfactory acuity; they appeared to have a better sense of smell. Thus, despite its striking expression pattern, OCAM is not essential for overall topographic OE-OB connectivity. Instead, OCAM is required for establishing or maintaining the compartmental organization and the segregation of axodendritic and dendrodendritic synapses within glomeruli.

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PubMed2006

Cytoskeletal organization of the developing mouse olfactory nerve layer.

Olfactory sensory neuron (OSN) axonal extension and targeting occur within the olfactory nerve layer (ONL) of the olfactory bulb (OB). The ONL can be differentiated into sublaminae: the outer (ONLo), where axons broadly target regions of the OB in tight fascicles, and inner (ONLi), where axons perform final targeting in loosely organized fascicles. During perinatal development, cadherin-2 and its binding partner, gamma-catenin, are preferentially expressed by OSN axons in the ONLo vs. the ONLi. Given the expression of these cytoskeleton-associated molecules, we hypothesized that cytoskeletal elements of OSN axons may be differentially expressed across the ONL. We therefore examined cytoskeletal organization of OSN axons in the ONL, focusing on the day of birth (P0). We show that microfilaments, microtubules, and the intermediate filament (IF) vimentin are homogeneously expressed across the ONL at P0. In contrast, the IFs peripherin and alpha-internexin are preferentially localized to the ONLo at P0, with alpha-internexin expressed by a restricted subset of OSNs. We also show that OSN axons in the ONLo are significantly smaller than those in the ONLi. The data demonstrate that, as OSN axons begin to exit the ONLo and target a specific region of the OB, there is a down-regulation of cytoskeletal elements and bound extracellular adhesion molecules. The increase in axon diameter may reflect additional mechanisms involved in glomerular targeting or the formation of the large terminal boutons of OSN axons within glomeruli.

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PubMed2006

Dendritic excitability and calcium signalling in the mitral cell distal glomerular tuft.

The processing of odour information starts at the level of the olfactory glomerulus, where the mitral cell distal dendritic tuft not only receives olfactory nerve sensory input but also generates dendrodendritic output to form complicated glomerular synaptic circuits. Analysing the membrane properties and calcium signalling mechanisms in these tiny dendritic branches is crucial for understanding how the glomerular tuft transmits and processes olfactory signals. With the use of two-photon Ca2+ imaging in rat olfactory bulb slices, we found that these distal dendritic branches displayed a significantly larger Ca2+ signal than the soma and primary dendrite trunk. A back-propagating action potential was able to trigger a Ca2+ increase throughout the entire glomerular tuft, indicative of the presence of voltage-gated Ca2+ conductances in all branches at different levels of ramification. In response to a train of action potentials evoked at 60 Hz from the soma, the tuft Ca2+ signal increased linearly with the number of action potentials, suggesting that these glomerular branches were able to support repetitive penetration of Na+ action potentials. When a strong olfactory nerve excitatory input was paired with an inhibition from mitral cell basal dendrites, a small spike-like fast prepotential was revealed at both the soma and distal primary dendrite trunk. Corresponding to this fast prepotential was a Ca2+ increase confined locally within the glomerular tuft. In summary, the mitral cell distal dendritic tuft possesses both Na+ and Ca2+ voltage-dependent conductances which can mediate glomerular Ca2+ responsiveness critical for dendrodendritic output and synaptic plasticity.

Action Potentials↗
PubMed2005

Expression of the neuronal calcium sensor protein NCS-1 in the developing mouse olfactory pathway.

Neuron specific calcium sensor 1 (NCS-1) is widely expressed in the developing and adult nervous system. Like calmodulin, NCS-1 is a member of a family of calcium binding proteins that contain EF-hand motifs, which bind calcium and induce conformational changes in the protein. Their binding varies with calcium concentration, allowing them to act as true calcium sensors rather than just calcium binding proteins. This family of proteins has been implicated in important synaptic events including neurotransmitter release and synapse formation. We examined the expression of NCS-1 in the developing and mature olfactory system to determine whether this molecule may be playing a role in establishing and/or maintaining olfactory circuitry. During development, expression of NCS-1 in the olfactory epithelium was localized in the dendritic knobs and axons of olfactory sensory neurons. Axonal expression was down-regulated after synapse formation. In the developing olfactory bulb, NCS-1 was expressed in the processes of mitral/tufted and granule cells. However, in the adult olfactory bulb, strongest expression was found in a subset of periglomerular cells (PGCs). This subset of PGCs did not express other known markers of PGCs including tyrosine hydroxylase, glutamic acid decarboxylase, calbindin, or calretinin, and only partially overlapped with the subpopulation of PGCs that express parvalbumin. Together, these data suggest multiple and overlapping roles of NCS-1 in the developing and mature olfactory system.

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PubMed2005

The alpha6 integrin subunit in the developing mouse olfactory bulb.

Integrins are heterodimeric cell surface receptors that mediate developmental events by binding extracellular matrix ligands. Several lines of evidence suggest a role for integrins, specifically the alpha 6 subunit, in neuronal migration, neurite outgrowth, and axon guidance during olfactory development. Therefore, we undertook an analysis of the expression of the alpha 6 subunit in the olfactory system of the embryonic and early postnatal mouse to understand the role it may play during neural development. In addition, as a functional assay we examined the developmental effects of the loss of this subunit on olfactory development by analyzing an alpha 6 knockout (alpha 6-/-). Immunohistochemical analyses and confocal microscopy were used to examine alpha 6 expression in the CD-1 embryonic and early postnatal olfactory system and also to examine the organization of the olfactory system in the alpha 6-/- mouse. In CD-1 mice from E13 to E17, alpha 6 localizes in radial patterns extending from the core of the olfactory bulb to the nerve layer and colocalizes with RC2, an antibody specific for radial glia. By the day of birth (P0; approximately E19), expression is limited to the external plexiform layer and the olfactory nerve layer, where it colocalizes with laminin and p75. In the alpha 6-/- mouse, areas of ectopic granule cells were observed in the mitral cell layer of the olfactory bulb. These ectopias coincided with areas of disorganization of the radial glial processes and breaks in the mitral cell layer. These observations suggest a role for alpha 6 integrin in neural migration during olfactory development, likely secondary to organization of the radial glial scaffold.

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PubMed2004

Nestin promoter/enhancer directs transgene expression to precursors of adult generated periglomerular neurons.

The subventricular zone (SVZ) is a major neurogenic region in the adult brain. Cells from the SVZ give rise to two populations of olfactory bulb interneurons: the granule cells and periglomerular (PG) cells. Currently, little is known about the signaling pathways that direct these newly generated neurons to become either granule or PG neurons. In the present study, we used the nestin promoter and enhancer to direct expression of the tetracycline transactivator (tTA). We generated two independent strains of nestin-tTA transgenic animals and crossed founder mice from both lines to mice containing a tetracycline-regulated transgene (mCREB) whose expression served as a marker for the activity of the nestin-tTA transgene. mCREB expression occurred in a subset of proliferating cells in the SVZ and rostral migratory stream in both lines. Surprisingly, in both lines of nestin-tTA mice transgene expression in the olfactory bulb was limited to PG neurons and was absent from granule cells, suggesting that this nestin promoter construct differentiates between the two interneuronal populations. Transgene expression occurred in several subtypes of PG neurons, including those expressing calretinin, calbindin, GAD67, and tyrosine hydroxylase. These results suggest that a unique subset of SVZ precursor cells gives rise to PG, and not granule cells. The ability to express different transgenes within this subpopulation of neuronal precursors provides a powerful system to define the signals regulating the differentiation and survival of adult-generated neurons in the olfactory bulb.

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PubMed2004

Postnatal refinement of peripheral olfactory projections.

Axonal projections from the olfactory epithelium to the olfactory bulb are organized into glomeruli according to the expressed odorant receptor. Using gene-targeted mice, we show that glomerular maturation proceeds along different time courses for two similar receptors and requires sensory input during distinct sensitive periods. During early development, some glomeruli are innervated by axons of neurons that do not express the same receptor. These heterogeneous glomeruli normally disappear with age, but they persist in adults deprived of sensory input by unilateral and permanent naris closure. Persistence may be due, in part, to prolonged survival of olfactory sensory neurons.

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PubMed2004

Remyelination of spinal cord axons by olfactory ensheathing cells and Schwann cells derived from a transgenic rat expressing alkaline phosphatase marker gene.

Transplantation of cell suspensions containing olfactory ensheathing cells (OECs) has been reported to remyelinate demyelinated axons in the spinal cord with a Schwann cell (SC)-like pattern of myelination. However, questions have been raised recently as to whether OECs can form SC-like myelin. To address this issue we prepared SCs and OECs from transgenic rats in which a marker gene, human placental alkaline phosphatase (hPAP), is linked to the ubiquitously active promoter of the R26 gene. SCs were prepared from the sciatic nerve and OECs from the outer nerve-fiber layer of the olfactory bulb. Positive S100 and p75 immunostaining indicated that >95% of cells in culture displayed either SC or OEC phenotypes. Suspensions of either SCs or OECs were transplanted into an X-irradiation/ethidium bromide demyelinating lesion in the spinal cord. We observed extensive SC-like remyelination following either SC or OEC transplantation 3 weeks after injection of the cells. Alkaline phosphatase (ALP) chromagen reaction product was associated clearly with the myelin-forming cells. Thus, cell suspensions that are enriched in either SCs or OECs result in peripheral-like myelin when transplanted in vivo.

Journal Article↗
PubMed2003

Cell surface carbohydrates and glomerular targeting of olfactory sensory neuron axons in the mouse.

Cell surface carbohydrates have been implicated in axon guidance and targeting throughout the nervous system. We have begun to test the hypothesis that, in the olfactory system, a differential distribution of cell surface carbohydrates may influence olfactory sensory neuron (OSN) axon targeting. Specifically, we have examined the spatial distribution of two different plant lectins, Ulex europaeus agglutinin (UEA) and Dolichos biflorus agglutinin (DBA), to determine whether they exhibit differential and reproducible projections onto the main olfactory bulb. Each lectin exhibited a unique spatial domain of glomerular labeling that was consistent across animals. UEA labeling was strongest in the ventral aspect of the olfactory bulb; DBA labeling was strongest in the dorsal aspect of the olfactory bulb. Some evidence for colocalization was present where these two borders intersected. Large areas of the glomerular layer were not labeled by either lectin. To determine whether patterns of lectin labeling were reproducible at the level of individual glomeruli, UEA labeling was assessed relative to M72-IRES-taulacZ- and P2-IRES-taulacZ-labeled axons. Although glomeruli neighboring these two identified glomeruli were consistently labeled with UEA, none of the lacZ positive axons was lectin labeled. Labeling of vomeronasal sensory neuron axons in the accessory olfactory bulb was more uniform for the two lectins. These data are the first to show a differential distribution of UEA vs. DBA labeling in the main olfactory bulb and are consistent with the hypothesis that a differential distribution of cell surface carbohydrates, a glycocode, may contribute to the targeting of OSN axons.

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PubMed2003

Glomerular territories in the olfactory bulb from the larval stage of the sea lamprey Petromyzon marinus.

The goal of this study was to investigate the spatial organization of olfactory glomeruli and of substances relevant to olfactory sensory neuron activity in the developing agnathan, the sea lamprey Petromyzon marinus. A 45-kD protein immunoreactive to G(olf), a cAMP-dependent olfactory G protein, was present in the ciliary fraction of sea lamprey olfactory epithelium and in olfactory sensory neurons of larval and adult sea lampreys. This result implies that G(olf) expression was present during early vertebrate evolution or evolved in parallel in gnathostome and agnathostome vertebrates. Serial sectioning of the olfactory bulb revealed a consistent pattern of olfactory glomeruli stained by GS1B(4) lectin and by anterograde labeling with fluorescent dextran. These glomerular territories included the dorsal cluster, dorsal ring, anterior plexus, lateral chain, medial glomeruli, ventral ring, and ventral cluster. The dorsal, anterior, lateral, and ventral glomeruli contained olfactory sensory axon terminals that were G(olf)-immunoreactive. However, a specific subset, the medial glomeruli, did not display this immunoreactivity. Olfactory glomeruli in the dorsal hemisphere of the olfactory bulb, the dorsal cluster, dorsal ring, anterior plexus, lateral chain, and medial glomeruli, were seen adjacent to 5HT-immunoreactive fibers. However, glomeruli in the ventral hemisphere, the ventral ring, and ventral cluster did not display this association. The presence of specific glomerular territories and discrete glomerular subsets with substances relevant to olfactory sensory neuron activity suggest a spatial organization of information flow in the lamprey olfactory pathway.

Animals↗
PubMed2003

Inverse expression of olfactory cell adhesion molecule in a subset of olfactory axons and a subset of mitral/tufted cells in the developing rat main olfactory bulb.

The projection of olfactory sensory neuron (OSN) axons from the olfactory epithelium (OE) to the olfactory bulb (OB) is highly organized but topographically complex. Evidence suggests that odorant receptor expression zones in the OE map to the OB about orthogonal axes. One candidate molecule for the formation of zone-specific targeting of OSN axon synapses onto the OB is the olfactory cell adhesion molecule (OCAM). OCAM(+) OSNs are restricted to three of the four zones in the OE and project their axons to the ventral OB where they form synapses with mitral/tufted (M/T) cells. To determine when this zonal connection is established, we have examined OCAM expression in rat olfactory system, during seminal periods of glomerular formation. OCAM(+) axons sort out in the ventral olfactory nerve layer of the OB before glomerular formation. Surprisingly, OCAM was also expressed transiently by subsets of M/T cell dendrites located in the dorsal OB. The expression of OCAM by OSN axons and M/T dendrites was asymmetrical; in the dorsal OB, OCAM(-) OSN axons synapsed on OCAM(+) M/T dendrites, whereas in the ventral OB, OCAM(+) OSN axons synapsed on OCAM(-) M/T dendrites. The restricted spatial map of OCAM(+) M/T cells appeared earlier in development than the zonal segregation of OCAM(+) OSN axons. Thus, OCAM on M/T cell dendrites may act in a spatiotemporal window to specify regions of the developing rat OB, thereby establishing a foundation for mapping of the OE zonal organization onto the OB.

Animals↗
PubMed2003

Olfactory signal transduction in the mouse septal organ.

The septal organ, a distinct chemosensory organ observed in the mammalian nose, is essentially a small island of olfactory neuroepithelium located bilaterally at the ventral base of the nasal septum. Virtually nothing is known about its physiological properties and function. To understand the nature of the sensory neurons in this area, we studied the mechanisms underlying olfactory signal transduction in these neurons. The majority of the sensory neurons in the septal organ express olfactory-specific G-protein and adenylyl cyclase type III, suggesting that the cAMP signaling pathway plays a critical role in the septal organ as in the main olfactory epithelium (MOE). This is further supported by patch-clamp recordings from individual dendritic knobs of the sensory neurons in the septal organ. Odorant responses can be mimicked by an adenylyl cyclase activator and a phosphodiesterase inhibitor, and these responses can be blocked by an adenylyl cyclase inhibitor. There is a small subset of cells in the septal organ expressing a cGMP-stimulated phosphodiesterase (phosphodiesterase 2), a marker for the guanylyl cyclase-D subtype sensory neurons identified in the MOE. The results indicate that the septal organ resembles the MOE in major olfactory signal transduction pathways, odorant response properties, and projection to the main olfactory bulb. Molecular and functional analysis of the septal organ, which constitutes approximately 1% of the olfactory epithelium, will provide new insights into the organization of the mammalian olfactory system and the unique function this enigmatic organ may serve.

Adenylyl Cyclases↗
PubMed2002

Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact.

Axon regeneration in the adult CNS is limited by the presence of inhibitory proteins. An interaction of Nogo on the oligodendrocyte surface with Nogo-66 Receptor (NgR) on axons has been suggested to play an important role in limiting axonal growth. Here, we compare the localization of these two proteins immunohistochemically as a test of this hypothesis. Throughout much of the adult CNS, Nogo-A is detected on oligodendrocyte processes surrounding myelinated axons, including areas of axon-oligodendrocyte contact. The NgR protein is detected selectively in neurons and is present throughout axons, indicating that Nogo-A and its receptor are juxtaposed along the course of myelinated fibers. NgR protein expression is restricted to postnatal neurons and their axons. In contrast, Nogo-A is observed in myelinating oligodendrocytes, embryonic muscle, and neurons, suggesting that Nogo-A has additional physiologic roles unrelated to NgR binding. After spinal cord injury, Nogo-A is upregulated to a moderate degree, whereas NgR levels are maintained at constant levels. Taken together, these data confirm the apposition of Nogo ligand and NgR receptor in situations of limited axonal regeneration and support the hypothesis that this system regulates CNS axonal plasticity and recovery from injury.

Animals↗
PubMed2002

Sublaminar organization of the mouse olfactory bulb nerve layer.

Olfactory sensory neuron (OSN) axons coalesce to form the olfactory nerve (ON) and then grow from the olfactory epithelium to the olfactory bulb (OB), enter the olfactory nerve layer (ONL), reorganize extensively, and innervate specific glomeruli. Within the ON and ONL a population of glial cells, the olfactory ensheathing cells (OECs), surround OSN axon fascicles. To better understand the relationship between OECs and axon fascicles in the ONL of the adult mouse, we used confocal microscopy and antibodies to the low affinity nerve growth factor receptor p75 (p75), glial fibrillary acidic protein (GFAP), neuropeptide Y (NPY), and S-100 to identify glia. Antibodies to olfactory marker protein (OMP) and neuronal cell adhesion molecule (NCAM) were used to identify OSN axons. Electron microscopy characterized the ONL ultrastructure. We found that glial processes were not uniformly distributed in the ONL of the mouse. The p75(+) OEC processes were restricted to the ON and the outer ONL sublamina, and oriented parallel to the plane of the OB layers. In the inner ONL NPY(+) OEC-like processes were seen. GFAP(+) processes were restricted to the inner ONL sublamina, the ONL/GL boundary, and the GL, where they delineated loosely aggregated axon fascicles that entered the glomeruli obliquely. S-100(+) processes and somata were distributed throughout the ONL; the outer and inner ONL were equivalent in their S-100 staining. Ultrastructural studies showed that, although OECs could be identified in both the outer and inner ONL, in the latter, their relationship to bundles of OEC axons appeared less orderly than seen in the outer ONL. Our data demonstrate a differential organization of the ONL that could subserve distinct functions; axon extension may occur predominantly in the outermost ONL, whereas glomerular targeting occurs in the inner sublamina of the ONL.

Animals↗
PubMed2002

Specificity of glomerular targeting by olfactory sensory axons.

Axons from olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) project to specific subsets of glomeruli in the olfactory bulb (for review, see Mombaerts, 1999, 2001). The aim of this study was to examine the trajectories that subsets of axons from OSNs expressing the same OR follow within the olfactory nerve and olfactory nerve layer (ONL) of adult mice. Using confocal microscopy, we generated serial reconstructions of axons from M72-IRES-tauGFP-expressing OSNs as they coursed within the ONL and into glomeruli. GFP-expressing axons were loosely aggregated in the outer ONL; however, as they entered the inner ONL, the majority fasciculated with other GFP-expressing axons before entering the glomerular neuropil. Although the vast majority of axons entered the glomerulus from the directly apposed ONL, some followed tortuous courses through and/or around adjacent glomeruli before terminating in the target glomerulus. Similar observations were made on subpopulations of axons in M71-IRES-tauGFP and P2-IRES-tauGFP mice. Ultrastructural analyses of labeled M72 glomeruli showed no evidence of axodendritic synapses other than those with GFP-labeled axon terminals. These data are consistent with the notion that OSN axons are highly precise in targeting glomeruli and that glomeruli, in turn, are highly homogeneous with regard to the OR expressed by the innervating OSNs. Because some single axons could follow idiosyncratic trajectories to the target glomerulus, it appears that stable homotypic fasciculation is not a prerequisite for correct targeting.

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