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C A Greer

Publications and source records attributed to C A Greer.

At least 19 recordsLinked to original sources
PubMed2001

Mechanisms of axonal plasticity: lessons from the olfactory pathway.

The olfactory pathway has emerged recently as an effective model for studying general principles of axon extension and regeneration. A variety of both trophic as well as repulsive molecules are found in the olfactory pathway and are being characterized for their roles in promoting the high capacity for plasticity and growth in olfactory receptor cell axons. In addition, olfactory ensheathing cells, which line the olfactory nerve, have been shown to promote axon extension not only in the olfactory pathway but also in the injured spinal cord. This review summarizes some of our current knowledge of these mechanisms and how they may function collectively to promote axon plasticity.

Animals↗
PubMed2000

The emergence of compartmental organization in olfactory bulb glomeruli during postnatal development.

The olfactory bulb glomerulus is a discrete and heterogeneous neuropil where olfactory receptor cell axons synapse with dendrites of mitral, tufted, and periglomerular neurons. To understand better the maturation of glomeruli and the spatiotemporal interactions that occur during postnatal development, we employed confocal microscopy and markers for immature and mature olfactory receptor cell axons in parallel with a marker for synaptic structure in maturing glomeruli. Sprague-Dawley rats at postnatal days 1, 6, 12, and 18 were processed for single- and double-label immunocytochemistry for olfactory marker protein (OMP), growth-associated protein (GAP-43), and synaptophysin. Mature or adult-like subcompartmental organization within the glomerulus emerged by postnatal day 12. Earlier in development immature axons entered the core of the glomerulus and moved to the periphery as they matured. However, beginning around 12 days postnatal, immature axons distributed in the periphery and moved toward the core as they matured. This change in the trajectories of axons into glomeruli suggests that different rules may be followed in establishing versus maintaining glomeruli. Double labeling with OMP and synaptophysin demonstrated strong colocalization compared with GAP-43 and synaptophysin, which showed much less colocalization, consistent with the notion that OMP is associated with more mature axons.

Age Factors↗
PubMed2000

Ultrastructural characteristics and conduction velocity of olfactory receptor neuron axons in the olfactory marker protein-null mouse.

Olfactory receptor neuron (ORN) axon diameters and the conduction velocity of the compound action potential along ORN axons were studied in olfactory marker protein (OMP)-null mice and genotypically matched controls. The compound action potential was distinguished from postsynaptic field potentials by its shorter latency, its persistence following application of cobalt or kynurenic acid that blocked postsynaptic responses, and its ability to follow paired-pulse stimulation at 300 Hz. Blockade of the postsynaptic field responses by kynurenic acid indicates that in the mouse, as in the rat, glutamate is the olfactory nerve transmitter. The mean conduction velocity of ORNs in wild-type control mice was 0. 47+/-0.19 (S.E.M.) m/s (n=5), similar to the conduction velocity reported for other mammals. The mean diameter of ORN axons in control mice was 0.202+/-0.005 and 0.261+/-0.006 microm in the OMP-null mice. This increase in fiber diameter in the OMP-nulls predicts an increase in impulse conduction velocity. However, the mean conduction velocity of OMP-null mice, 0.38+/-0.03 m/s (n=6), was not significantly different from control (P>0.1). The conduction velocity predicted by the increase in fiber diameter in OMP-null mice was within the 95% confidence interval of the measured value. Thus, OMP-null ORNs are normal with respect to the conduction velocity of their axons. The number of axodendritic synapses in the glomeruli of OMP-null mice is higher than in congenic wild-type mice.

Action Potentials↗
PubMed2000

Cyclic GMP evoked calcium transients in olfactory receptor cell growth cones.

Nitric oxide-induced calcium transients in growth cones are believed to be mediated by cyclic nucleotides. Because nitric oxide is thought to influence the development of olfactory receptor cells (ORCs), we have begun to explore the effect of cyclic nucleotides on ORC growth cones. Cultured ORCs were loaded with fluo-3 AM and confocal imaging was employed to monitor calcium transients following cyclic nucleotide-gated channel activation. Application of 8-bromo-cGMP at the growth cone caused transient increases in fluorescence which were restricted to the growth cone and lasted tens of seconds. The signal was abolished by LY83583, an inhibitor of cyclic nucleotide-gated channels. 8-Bromo-cGMP also inhibited further extension of growth cones. The data indicate that ORC growth cones exhibit cGMP-dependent calcium transients that are consistent with those generated by cyclic nucleotide-gated channels.

Animals↗
PubMed2000

A putative pheromone receptor gene expressed in human olfactory mucosa.

Pheromones elicit specific behavioural responses and physiological alterations in recipients of the same species. In mammals, these chemical signals are recognized within the nasal cavity by sensory neurons that express pheromone receptors. In rodents, these receptors are thought to be represented by two large multigene families, comprising the V1r and V2r genes, which encode seven-transmembrane proteins. Although pheromonal effects have been demonstrated in humans, V1R or V2R counterparts of the rodent genes have yet to be characterized.

Alleles↗
PubMed2000

Olfactory ensheathing cells: bridging the gap in spinal cord injury.

Spinal cord injury (SCI) continues to be an insidious and challenging problem for scientists and clinicians. Recent neuroscientific advances have changed the pessimistic notion that axons are not capable of significant extension after transection. The challenges of recovering from SCI have been broadly divided into four areas: 1) cell survival; 2) axon regeneration (growth); 3) correct targeting by growing axons; and 4) establishment of correct and functional synaptic appositions. After acute SCI, there seems to be a therapeutic window of opportunity within which the devastating consequences of the secondary injury can be ameliorated. This is supported by several observations in which apoptotic glial cells have been identified up to 1 week after acute SCI. Moreover, autopsy studies have identified anatomically preserved but unmyelinated axons that could potentially subserve normal physiological properties. These observations suggest that therapeutic strategies after SCI can be directed into two broad modalities: 1) prevention or amelioration of the secondary injury, and 2) restorative or regenerative interventions. Intraspinal transplants have been used after SCI as a means for restoring the severed neuraxis. Fetal cell transplants and, more recently, progenitor cells have been used to restore intraspinal circuitry or to serve as relay for damaged axons. In an attempt to remyelinate anatomically preserved but physiologically disrupted axons, newer therapeutic interventions have incorporated the transplantation of myelinating cells, such as Schwann cells, oligodendrocytes, and olfactory ensheathing cells. Of these cells, the olfactory ensheathing cells have become a more favorable candidate for extensive remyelination and axonal regeneration. Olfactory ensheathing cells are found along the full length of the olfactory nerve, from the basal lamina of the epithelium to the olfactory bulb, crossing the peripheral nervous system-central nervous system junction. In vitro, these cells promote robust axonal growth, in part through cell adhesion molecules and possibly by secretion of neurotrophic growth factors that support axonal elongation and extension. In animal models of SCI, transplantation of ensheathing cells supports axonal remyelination and extensive migration throughout the length of the spinal cord. Although the specific properties of these cells that govern enhanced axon regeneration remain to be elucidated, it seems certain that they will contribute to the establishment of new horizons in SCI research.

Acute Disease↗
PubMed2000

Amplification of odor-induced Ca(2+) transients by store-operated Ca(2+) release and its role in olfactory signal transduction.

A critical role of Ca(2+) in vertebrate olfactory receptor neurons (ORNs) is to couple odor-induced excitation to intracellular feedback pathways that are responsible for the regulation of the sensitivity of the sense of smell, but the role of intracellular Ca(2+) stores in this process remains unclear. Using confocal Ca(2+) imaging and perforated patch recording, we show that salamander ORNs contain a releasable pool of Ca(2+) that can be discharged at rest by the SERCA inhibitor thapsigargin and the ryanodine receptor agonist caffeine. The Ca(2+) stores are spatially restricted; emptying produces compartmentalized Ca(2+) release and capacitative-like Ca(2+) entry in the dendrite and soma but not in the cilia, the site of odor transduction. We deplete the stores to show that odor stimulation causes store-dependent Ca(2+) mobilization. This odor-induced Ca(2+) release does not seem to be necessary for generation of an immediate electrophysiological response, nor does it contribute significantly to the Ca(2+) transients in the olfactory cilia. Rather, it is important for amplifying the magnitude and duration of Ca(2+) transients in the dendrite and soma and is thus necessary for the spread of an odor-induced Ca(2+) wave from the cilia to the soma. We show that this amplification process depends on Ca(2+)-induced Ca(2+) release. The results indicate that stimulation of ORNs with odorants can produce Ca(2+) mobilization from intracellular stores without an immediate effect on the receptor potential. Odor-induced, store-dependent Ca(2+) mobilization may be part of a feedback pathway by which information is transferred from the distal dendrite of an ORN to its soma.

Animals↗
PubMed1999

G(o) protein-dependent survival of primary accessory olfactory neurons.

Extensive G protein-coupled receptor families in both the main and accessory olfactory systems have been implicated in axonal targeting, sensory function, and cell survival. Although sensory function seems to be mediated by G proteins, axonal guidance and cell survival may be G protein-independent processes. In the accessory olfactory system, the G(o)-containing neurons in the basal vomeronasal organ (VNO) project to the posterior accessory olfactory bulb (AOB), whereas more apically located VNO neurons contain G(i2) and project to the anterior AOB. Herein, we investigate the organization of the accessory olfactory system in mice with a targeted deletion in the G(o)alpha gene. The accessory olfactory system seems normal at birth; however, postnatally, the number of G(o)-receptor-containing VNO neurons decreases by half, and apoptotic neurons are detected. The axons of VNO neurons remain restricted to the posterior AOB. The posterior AOB is reduced in size but contains a synaptophysin-positive layer with the normal number of glomeruli. The posterior AOB has reduced mitral cell c-Fos immunoreactivity, consistent with decreased sensory activation of G(o) protein-coupled VNO receptor neurons. Thus, in the accessory olfactory system, receptor-coupled G proteins are required for cell survival.

Animals↗
PubMed1999

Glomerular formation in the developing rat olfactory bulb.

Using the confocal microscope together with markers for the cellular components of glomeruli, we examined the spatiotemporal cellular interactions that occur between the axons of olfactory receptor cells, their dendritic targets, and glial cells during the critical period of glomerular formation. We have employed markers of immature and mature olfactory receptor cell axons, mitral/tufted cell dendrites, and glial cells as well as a synapse-associated protein for double- and triple-label immunocytochemistry. Axons of olfactory receptor cells grew into a dense dendritic zone of the olfactory bulb (comprising the dendrites of both mitral and tufted cells) between E17 and E18. At E19, these axons coalesced into protoglomeruli, which continued to develop until birth, when the basic anatomical structure of adult glomeruli emerged. Neither mitral/tufted cell dendrites nor olfactory bulb astrocytes became specifically associated with these protoglomeruli until E21. Ensheathing cells remained restricted to the outer nerve fiber layer and did not appear to contribute to glomerular formation. Finally, the synaptophysin staining has shown that synaptic constituents are expressed as early as E17, prior to the appearance of mature olfactory receptor cell axons. Based on these data, we have established a time line detailing the temporal and spatial interactions that occur between cell types during late embryonic rat olfactory bulb development. We conclude that the initial event in the formation of glomeruli is the penetration of the mitral/tufted cell dendritic zone by olfactory receptor cell axons. The coalescence of dendritic and glial processes into glomerular structures appears secondary to the arrival of the olfactory receptor cell axons.

Animals↗
PubMed1999

Compartmental organization of the olfactory bulb glomerulus.

Olfactory receptor cell (ORC) axons terminate in the olfactory bulb glomerular neuropil, where they synapse with dendrites of mitral, tufted, and periglomerular neurons. We investigated the organization of the glomerular neuropil by using antibodies to both single- and double-label constituents for analyses with confocal microscopy. Electron microscopy (EM) was employed to assess the distribution of synaptic appositions within the glomerulus. Adult Sprague-Dawley rats were processed for immunocytochemistry with olfactory marker protein (OMP), synaptophysin, synapsin 1, glial fibrillary acidic protein (GFAP), and/or microtubule-associated protein 2 (MAP2). Equivalent rats were processed for transmission EM. Double labeling for OMP and MAP2 revealed two distinctive subcompartments within glomeruli: an axonal compartment containing predominately primary afferent axons with individual dendritic inserts and a complementary dendritic compartment that excluded primary afferent axons. Areas not occupied by OMP or MAP2 immunoreactivity were either immunoreactive for GFAP, indicating a glial process, or were blood vessels. Synaptophysin and synapsin 1 also showed differential labeling within the glomerulus. Synaptophysin strongly colocalized with OMP, whereas synapsin 1 was associated most strongly with MAP2. Reconstructions of glomeruli from EM montages revealed interdigitating axonal and dendritic subcompartments. The axonal subcompartments were composed primarily of ORC processes with individual or small groups of dendrites interspersed. Dendritic subcompartments were composed predominately of dendritic processes. Primary afferent axodendritic and local-circuit dendrodendritic synapses segregated within the glomerulus into the axonal and dendritic subcompartments, respectively. The results support the hypothesis of subcompartmental organization within olfactory bulb glomeruli.

Animals↗
PubMed1999

Differential distribution of ionotropic glutamate receptor subunits in the rat olfactory bulb.

The subcellular localization of ionotropic glutamate receptor (GluR) subunits was examined with light and electron microscopy in the rat olfactory bulb by using antibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits: GluR1, GluR2/3, and GluR4; and kainate (KA) receptor subunits: GluR5/6/7. Immunoreactivity to GluR1 was heavy in the glomerular layer, moderate in the external plexiform layer, and localized to periglomerular somata and dendrites, short axon somata and dendrites, mitral cell somata, and mitral/tufted dendrites. GluR2/3 immunoreactivity was heavy in the external plexiform and glomerular layers and localized to periglomerular somata and dendrites, mitral cell somata, mitral/tufted dendrites, granule cell somata, and olfactory nerve-associated glia. GluR4 immunoreactivity showed heavy staining in the external plexiform and olfactory nerve layers with localization to mitral cells, mitral/tufted dendritic processes, and olfactory nerve glial processes. GluR5/6/7 immunoreactivity was heavy in the external plexiform layer, moderate in the olfactory nerve and glomerular layers, and localized to granule cells, mitral cells, and mitral/tufted dendritic processes. Ultrastructural immunolabeling for all antibodies examined showed immunoreactivity in the postsynaptic membrane and densities, adjacent dendritic cytoplasm, and somatic cytoplasm. These data demonstrate a highly specific laminar, cellular, and subcellular distribution of ionotropic GluR subunits within the primary afferent and local synaptic circuits of the olfactory bulb. The results are consistent with the notion that the different roles subserved by glutamate in the olfactory bulb are actuated, in part, by a differential distribution of GluR subunits.

Animals↗
PubMed1999

Olfactory ensheathing cells promote neurite extension from embryonic olfactory receptor cells in vitro.

The role of ensheathing cells, a macroglial cell type with a unique presence in the olfactory system, in the outgrowth of olfactory receptor cell neurites was explored in vitro. Glial cell cultures harvested from both the olfactory bulb nerve layer and the hippocampus were established and immunocytochemically characterized. The expression of the p75 low-affinity nerve growth factor receptor by ensheathing cells was used to distinguish them from other macroglial subpopulations. Results indicated that ensheathing cell cultures were approximately 80% pure. Olfactory receptor cells were cocultured with ensheathing or hippocampal glial cells or were seeded on laminin or poly-L-lysine as controls. Olfactory receptor cells extended the longest primary neurites when cocultured with ensheathing cells. Neurite extension on hippocampal glia and laminin was less extensive than that observed on ensheathing cells but higher than that on poly-L-lysine. The neurite outgrowth-promoting effect of ensheathing cells was, at least in part, mediated by diffusible factors, because olfactory receptor cell neurite extension could also be facilitated when receptor cells were cultured in ensheathing cell-conditioned media. In contrast, cortical neurons extended neurites of equivalent lengths on ensheathing and hippocampal glia. The results suggest that ensheathing cells may release factors that support the continuous outgrowth of olfactory receptor cell axons and, therefore, the capacity of this pathway to recover from injury.

Animals↗
PubMed1998

Visualizing odor detection in olfactory cilia by calcium imaging.

To visualize odor detection in individual cilia of olfactory sensory neurons we have developed a new approach by using high-resolution calcium imaging techniques. Laser scanning confocal microscopy revealed, for the first time, that odor stimuli induce transient Ca2+ elevations in single olfactory cilia. Pharmacological analysis indicates that these Ca2+ signals depend entirely on Ca2+ entry through activated cyclic nucleotide-gated (CNG) channels. This novel approach enables us to monitor the initial steps leading to olfactory perception in a spatially and temporally resolved manner.

Animals↗
PubMed1998

The influence of ensheathing cells on olfactory receptor cell neurite outgrowth in vitro.

We previously reported that laminin substrates increased primary (1 degree) neurite outgrowth from olfactory receptor cells (ORCs) in vitro. To further explore mechanisms underlying the outgrowth of ORC neurites, we have cocultured ORCs with the ensheathing cells (ENSH) from the olfactory nerve. ORCs were plated either: (i) directly on monolayers of ENSH (prepared with minor modifications as reported by Doucette and Devon, or (ii) on coverslips suspended above the ENSH monolayer to investigate diffusible trophic influences of ENSH. In addition, ORCs were cocultured with either olfactory bulb glia (OBG) or hippocampal astrocytes (HG) or grown on either laminin (LN) substrates or poly-L-lysine (PLL) controls. The length of ORC neurites was determined after 48 hr in vitro. Immunocytochemical characterization of the ENSH cultures for p75 nerve growth factor (NGF) receptor and glial fibrillary acidic protein (GFAP) revealed that those cultures contained more than 80% ENSH. In OBG cultures approximately 10% and in HG cultures no cells with ENSH characteristics were found. All cells with ENSH characteristics were also LN-immunoreactive. After 48 hr in culture ORCs had the longest 1 degree neurites when they were cocultured with ENSH. No significant differences in the 1 degree neurite length were found comparing ORCs grown directly on ENSH and ORCs physically separated from ENSH. ORCs cultured on HG and on EHS-LN showed no significant differences in the ORC 1 degree neurite length, but on both substrates the ORC 1 degree neurites were significantly shorter than on ENSH. The length of the ORC secondary neurites did not vary significantly in the different culture conditions. Our results suggest that while LN appears to contribute to ORC neurite extension, additional diffusible factors released from ENSH are likely to be further determinants of neurite outgrowth. Because the OBG and HG cocultures did not influence ORC neurite outgrowth as significantly as did the ENSH, it seems plausible to suggest that these diffusible factors may be specific to subpopulations of glial cells.

Animals↗
PubMed1998

Identification of olfactory receptor mRNA sequences from the rat olfactory bulb glomerular layer.

In situ hybridization has demonstrated mRNA for olfactory receptors (OR) in the axon terminals of olfactory receptor neurons. Neurons that express the same OR appear to send their axons to two stereotyped glomeruli in the olfactory bulb (OB). Based on these observations, we tested the feasibility of using RT-PCR to isolate and sequence OR mRNA from small samples of the rat OB glomerular layer. Biomagnetic mRNA isolation followed by RT-PCR yielded partial sequences for 21 novel members of the OR family. The results suggest that the topography of OR mRNA can be mapped across the OB, to study synaptic specificity and odor representation in the olfactory system.

Animals↗
PubMed1998

Transplanted olfactory ensheathing cells remyelinate and enhance axonal conduction in the demyelinated dorsal columns of the rat spinal cord.

Olfactory ensheathing cells (OECs), which have properties of both astrocytes and Schwann cells, can remyelinate axons with a Schwann cell-like pattern of myelin. In this study the pattern and extent of remyelination and the electrophysiological properties of dorsal column axons were characterized after transplantation of OECs into a demyelinated rat spinal cord lesion. Dorsal columns of adult rat spinal cords were demyelinated by x-ray irradiation and focal injections of ethidium bromide. Cell suspensions of acutely dissociated OECs from neonatal rats were injected into the lesion 6 d after x-ray irradiation. At 21-25 d after transplantation of OECs, the spinal cords were maintained in an in vitro recording chamber to study the conduction properties of the axons. The remyelinated axons displayed improved conduction velocity and frequency-response properties, and action potentials were conducted a greater distance into the lesion, suggesting that conduction block was overcome. Quantitative histological analysis revealed remyelinated axons near and remote from the cell injection site, indicating extensive migration of OECs within the lesion. These data support the conclusion that transplantation of neonatal OECs results in quantitatively extensive and functional remyelination of demyelinated dorsal column axons.

Animals↗
PubMed1998

The expression of tenascin-C along the lamprey olfactory pathway during embryonic development and following axotomy-induced replacement of the olfactory receptor neurons.

Extracellular guidance molecules affect the pathway of growing axons by both attractive and repulsive interactions. Tenascin-C, a glycoprotein of the extracellular matrix, is localized along developing axonal pathways where it may function by repulsion, restricting axons within specific boundaries. The lamprey olfactory pathway offers an advantageous model for studying the role of extracellular matrix proteins in axon guidance because the entire pathway is readily seen in horizontal sections and because lesioning the olfactory nerve will induce the system into a new phase of coordinated neurogenesis and axon outgrowth. Although tenascin-C expression was absent during embryonic development, olfactory nerve fascicles contained tenascin-C-immunoreactivity (IR) during the larval stage. During retrograde degeneration, the fascicles lost tenascin-C-IR. Diffuse unfasciculated axonal processes extending from the olfactory epithelium did not express tenascin-C-IR; however, acetylated tubulin and GAP-43-IR was present, indicating axonal outgrowth. When the newly extended axons of olfactory receptor neurons converged to form fascicles, tenascin-C-IR was evident within the fascicular boundaries. The absence of tenascin-C expression when axonal process were short and diffuse, and its return when axons coalesced within fascicles, supports the view that tenascin-C functions as a boundary molecule in the olfactory pathway.

Animals↗