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C A Greer

Publications and source records attributed to C A Greer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Imaging odor-induced calcium transients in single olfactory cilia: specificity of activation and role in transduction.

The possibility that odor stimuli trigger distinct Ca2+ elevations within the cilia of vertebrate olfactory receptor neurons (ORNs) is a widely proposed concept. However, because of the small size of the olfactory cilia, the existence and properties of such Ca2+ elevations and their role in odor transduction are still unknown. We investigate odor-induced Ca2+ changes in individual olfactory cilia from salamander using the Ca2+ indicator dye fluo-3 in combination with laser scanning confocal microscopy. Single brief applications of odor ligand produce highly localized Ca2+ elevations in individual cilia lasting for several seconds. These Ca2+ signals originate in the cilia and depend entirely on Ca2+ entry through ciliary cyclic nucleotide-gated ion channels. The odor specificity of the Ca2+ rises implies a receptor-operated mechanism underlying odor detection. Each of the cilia on a receptor neuron functions as an independent biochemical compartment that can detect odorants and produce a Ca2+ transient with remarkably uniform properties in terms of kinetics and odor specificity. The rate of recovery of the odor-induced Ca2+ transients matches recovery from a short-term form of odor adaptation. Application of the membrane-permeant intracellular Ca2+ chelator BAPTA AM eliminates this odor adaptation. The results indicate that an olfactory cilium serves as a basic functional unit at the input level of the olfactory system, controlling both the specificity and sensitivity of odor detection.

Adaptation, Physiological↗
PubMed1998

Dynamic mapping at the laminar level of odor-elicited responses in rat olfactory bulb by functional MRI.

We have applied functional MRI (fMRI) based on blood oxygenation level-dependent (BOLD) image-contrast to map odor-elicited olfactory responses at the laminar level in the rat olfactory bulb (OB) elicited by iso-amyl acetate (10(-2) dilution of saturated vapor) with spatial and temporal resolutions of 220x220x1,000 micro(m) and 36 s. The laminar structure of the OB was clearly depicted by high-resolution in vivo anatomical MRI with spatial resolution of 110x110x1,000 micro(m). In repeated BOLD fMRI measurements, highly significant (P < 0.001) foci were located in the outer layers of both OBs. The occurrence of focal OB activity within a domain at the level of individual glomeruli or groups of glomeruli was corroborated on an intra- and inter-animal basis under anesthetized conditions with this noninvasive method. The dynamic studies demonstrated that the odor-elicited BOLD activations were highly reproducible on a time scale of minutes, whereas over tens of minutes the activations sometimes varied slowly. We found large BOLD signal (DeltaS/S = 10-30%) arising from the olfactory nerve layer, which is devoid of synapses and composed of unmyelinated fibers and glial cells. Our results support previous studies with other methods showing that odors elicit activity within glomerular layer domains in the mammalian OB, and extend the analysis to shorter time periods at the level of individual glomeruli or groups of glomeruli. With further improvement, BOLD fMRI should be ideal for systematic analysis of the functional significance of individual glomeruli in olfactory information encoding and of spatiotemporal processing within the olfactory system.

Animals↗
PubMed1998

Differential expression of extracellular matrix and cell adhesion molecules in the olfactory nerve and glomerular layers of adult rats.

Owing to the continual turnover of afferent input, the olfactory system offers a unique opportunity to study development and reorganization of neuronal networks in adults. To explore substrates that may underlie these processes in the adult olfactory system, we examined the expression and distribution of extracellular matrix and cell adhesion molecules (CAM) thought to be involved in axon guidance/extension. N-CAM, laminin, and tenascin were all detected by immunocytochemistry in the nerve and glomerular layers of the adult rat olfactory bulb, although the intensity and laminar distribution were varied. Antisera for N-CAM(total), N-CAM180, and tenascin bound to fascicles within the olfactory nerve layer and the glomerular neuropil. However, binding was nonuniform in that only subsets of axon fascicles and restricted glomeruli showed evidence of immunoreactivity. Antilaminin and a polyclonal antitenascin similarly exhibited heterogeneous intralaminar immunoreactivity. Tenascin colocalized with glial processes at the borders of glomeruli and subcompartments of the glomerular neuropil. Laminin immunoreactivity was evident in subsets of olfactory nerve fascicles and, to a lesser extent, the glomeruli. The data are consistent with the notion that ongoing axon extension and glomerular targeting in the olfactory system is subserved in part by a heterogeneous expression of the same extracellular matrix and CAMs present at higher levels during perinatal development.

Animals↗
PubMed1998

Postnatal development of olfactory receptor cell axonal arbors.

The mechanisms that subserve the distribution of the terminal arbors of olfactory receptor cell axons remain unknown. Elsewhere in the central nervous system, a common theme is early axonal exuberance followed by activity-dependent pruning to achieve the mature distribution. This led to the hypothesis that the orderly morphology of afferent axons in the olfactory glomerulus may follow a similar developmental scheme of exuberance followed by pruning. To test this hypothesis, we studied morphological features of olfactory receptor neuron (ORN) axonal arbors on postnatal days 0, 3, 6, 9, 12, and 21. The olfactory bulbs from Sprague-Dawley rats were processed using a Golgi technique that impregnated ORN axons. Axons from each age group were reconstructed by using camera lucida at x100, oil immersion, and morphometrically characterized. In the adult, the percent glomerular area occupied by a single ORN axon was 14%, whereas the mean length of branches was 169.67 microm, the sum of branches and varicosities was 27, and the distance to first branch point in glomeruli was 21.98 microm. The values from the younger age groups were not statistically different from those in the adult. Because there was no evidence of early exuberance, our data suggest that ORN axons must innervate single glomeruli and arborize in a highly specific manner to achieve the adult pattern. Because our data suggest that ORN axons do not follow the hypothesized scheme, it is plausible to suggest that as ORN axons innervate a glomerulus during development they arborize to their adult levels but not beyond. This argues strongly that specific cell surface and trophic factors are used by the ORN axon to guide glomerular targeting and innervation.

Animals↗
PubMed1998

The organization of piriform cortex and the lateral olfactory tract following the loss of mitral cells in PCD mice.

Homozygous Purkinje Cell Degeneration (PCD) mice exhibit a selective loss of olfactory bulb mitral cells (MCs) after 4 months of age. This selective degeneration leaves a subpopulation of denervated granule cells which establish new reciprocal dendro-dendritic synapses with unaffected tufted cells (TCs) (14). This suggests a capacity for plasticity in TCs and raises the question of whether a comparable degree of reorganization occurs in their axonal terminals in piriform cortex (PC) following the loss of MCs. Homozygous (experimental) and heterozygous (control) PCD mice were routinely perfused and processed for electron microscopy. A quantitative electron microscopic analysis was performed on radially oriented micrograph montages spanning from the pia into layer II of PC. After MC loss in the experimental animals there was a decrease in density of larger myelinated axons in the lateral olfactory tract (LOT). Myelinated axons in the LOT had a mean cross-sectional diameter of 1.26 +/- 0.04, and 0.81 +/- 0.025 microm in the control and experimental mice, respectively. In superficial layer I of PC, control mice had presynaptic axonal terminals from mitral and tufted cells with characteristic electron lucent (light) profiles establishing asymmetric synapses with pyramidal cell dendrites. In contrast, the experimental mice showed a decrease in electron lucent terminals and a robust increase in electron dense (dark) presynaptic associational terminals. Although the overall synaptic density did not differ between the control and experimental mice (16.40 +/- 0.94 and 18.10 +/- 0.96 synapses/100 microm2, respectively), an overall decrease in the thickness of Layer 1 suggests that the total number of synapses decreases following MC loss. In addition to the apparent increase of associational terminals, the diameter of terminal enlargements increased as well as the number of multiple synaptic contact per terminals in the experimental animal, suggesting further compensatory mechanisms for the loss of MC presynaptic terminals.

Animals↗
PubMed1997

Calcium entry through cyclic nucleotide-gated channels in individual cilia of olfactory receptor cells: spatiotemporal dynamics.

Transient elevations of intracellular Ca2+ play an important role in regulating the sensitivity of olfactory transduction, but such elevations have not been demonstrated in the olfactory cilia, which are the site of primary odor transduction. To begin to understand Ca2+ signaling in olfactory cilia, we used high-resolution imaging techniques to study the Ca2+ transients that occur in salamander olfactory receptor neurons (ORNs) as a result of cyclic nucleotide-gated (CNG) channel activation. To visualize ciliary Ca2+ signals, we loaded ORNs with the Ca2+ indicator dye Fluo-3 AM and measured fluorescence with a laser scanning confocal microscope. Application of the phosphodiesterase inhibitor IBMX increased fluorescence in the cilia and other neuronal compartments; the ciliary signal occurred first and was more transient. This signal could be abolished by lowering external Ca2+ or by applying LY83583, a potent blocker of CNG channels, indicating that Ca2+ entry through CNG channels was the primary source of fluorescence increases. Direct activation of CNG channels with low levels of 8-Br-cGMP (1 microM) led to tonic Ca2+ signals that were restricted locally to the cilia and the dendritic knob. Elevated external K+, which depolarizes cell membranes, increased fluorescence signals in the cell body and dendrite but failed to increase ciliary Ca2+ fluorescence. The results demonstrate the existence and spatiotemporal properties of Ca2+ transients in individual olfactory cilia and implicate CNG channels as a major pathway for Ca2+ entry into ORN cilia during odor transduction.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Downregulation of Na+ channel mRNA in olfactory bulb tufted cells following deafferentation.

Unilateral naris cauterization blocks odorant access to ipsilateral olfactory receptor cells and results in functional deafferentation of the olfactory bulb (OB). We used naris cauterization on postnatal day 2 (P2) to study the effects of deafferentation on the expression of Na+ channel subunits in OB. In situ hybridization at P18 showed that expression of Na+ channel alpha II and beta I mRNA was dramatically downregulated in tufted cells, while signals in mitral cells showed no detectable changes. Our observations suggest that during critical periods of development in some neurons, Na+ channel expression may be modulated by physiological activity. The differential response in the tufted and mitral cells may reflect varying degrees of dependence on afferent input or fundamental differences in cell properties.

Afferent Pathways↗
PubMed1997

Role of laminin in axonal extension from olfactory receptor cells.

The role of laminin, an extracellular matrix molecule believed to be involved in axon extension, was explored in the outgrowth of olfactory receptor cells and therefore in the maintenance of organization in the olfactory pathway. First, immunocytochemistry was used to examine laminin expression in the olfactory nerve and bulb during development. Laminin immunoreactivity was high in the olfactory nerve and glomerular layers. Although it declined in intensity, laminin expression continued in the nerve and in single glomeruli of adults. Second, the influence of laminin on neurite outgrowth was examined in vitro using olfactory receptor cells harvested from E14 rat embryos. We developed an in vitro assay to quantify the substrate preference of outgrowing neurites. Cells were cultured for 48 h on coverslips coated with either poly-L-lysine alone, or poly-L-lysine overlaid with laminin. On laminin-coated regions of coverslips, the primary neurites of olfactory receptor cells were 52% longer than on the poly-L-lysine control substrates. In addition, the direction of the neurite outgrowth was influenced by laminin. Fifty-six percent of all receptor cells located in a defined area surrounding a laminin zone extended neurites onto laminin. In contrast, only 7% of all receptor cells located in the corresponding laminin zone extended a neurite onto poly-L-lysine. In summary, these data suggest that laminin provides a favorable substrate for the extension of the primary neurite from olfactory receptor cells and the direction of their extension. Therefore, laminin may be a factor underlying continuous olfactory receptor cell axon outgrowth and its pathfinding in the olfactory system.

Animals↗
PubMed1996

Immunocytochemical analyses of astrocyte development in the olfactory bulb.

Several lines of evidence suggest an important role for glia in establishing boundaries during development of mammalian cortex and insect olfactory lobe. In the adult rat olfactory bulb distinct morphological categories of astroglial cells with clear laminar specificity are easily recognized following immunocytochemical staining of glial fibrillary acidic protein (GFAP). To explore the developmental distribution of olfactory bulb astrocytes and their possible role in establishing the segregation of neurons in specific olfactory bulb laminae, we used immunocytochemical localization of GFAP in rats at 0, 6, 9, 12, 15 and 21 days postnatal plus the adult. In the adult we confirmed prior observations and identified five morphological categories of astrocytes: linear, wedge, elongate, semicircular, and circular. Each category had a unique sublaminar distribution across the olfactory bulb, although categories could occur in more than one lamina. Between 0 and 21 days postnatal a 6th category was apparent, radial glial cells. The mature astrocyte morphologies did not emerge uniformly. Astrocytes found in the outermost glomerular layer developed first with the appearance of the linear, wedge and elongate morphologies. Deeper laminate of the olfactory bulb followed in a successive fashion until the adult pattern was evident around 15 days postnatal. As radial glia disappeared, the mature morphologies assumed their final position. The data suggest that the maturation of olfactory bulb astrocytes may be linked to the final migration and maturation of olfactory bulb neurons.

Animals↗
PubMed1996

Cell-specific differential expression of Na(+)-channel beta 1-subunit mRNA in the olfactory system during postnatal development and after denervation.

Activity-dependent mechanisms have been implicated in olfactory system development but, although such activity requires ion channels, few reports have described their expression in the olfactory system. We investigated the developmental and denervation-induced regulation of the Na(+)-channel beta 1 subunit (Na beta 1) in rat olfactory bulb (OB) and piriform cortex (PC). In situ hybridization shows that Na beta 1 mRNA expression is upregulated developmentally, but with different time courses in mitral, tufted, and pyramidal cells. In mitral cells, label was detected at postnatal day 4 (P4) and gradually increased to P14. Tufted cells were devoid of Na beta 1 mRNA before P14, when most cells expressed adult levels. In pyramidal cells of PC, Na beta 1 expression was not detectable clearly until P14, with maximal expression at P28. To examine the regulation of Na beta 1 mRNA, we surgically deafferented the OB at P30 and compared the effects on Na beta 1 with those for Na(+)-channel alpha-subunit (Na alpha) mRNAs. Within 5 d of surgery, the Na beta 1 and Na alpha II signals within tufted cells disappeared almost completely. Na beta 1 and Na alpha II expression was decreased in mitral cells to low-to-moderate levels. In pyramidal cells, Na beta 1 mRNA expression was decreased moderately without significant changes in Na alpha II mRNA. Deafferentation had no detectable effects on Na alpha I or III mRNAs in either OB or PC. These data indicate that Na beta 1 mRNA is expressed differentially in subpopulations of cells in the olfactory system during development and after deafferentation and suggest that the expression of Na beta 1 is regulated independently of Na alpha mRNAs via cell-specific and pathway-specific mechanisms.

Animals↗
PubMed1994

NADPH-diaphorase localization in the olfactory system.

NADPH-diaphorase staining was studied in the intact olfactory system and following transections of the olfactory nerve. Intense staining was found in the olfactory epithelia of rat and salamander, especially at the mucosal surface. The olfactory bulb nerve and glomerular layers were also stained. Transection of the olfactory nerve in salamander, or bulbectomy in rat, resulted in decreased staining at the mucosal surface, although staining increased in cells deeper in the epithelium. Deafferentation also caused the disappearance of staining in the olfactory nerve and glomerular layers of the bulb. These results support the notion of a role for NADPH in the olfactory system, perhaps as a biosynthesis substrate for NO modulation of cGMP in the developing epithelium and a complementary role for CO in the adult.

Animals↗
PubMed1994

Local communication within dendritic spines: models of second messenger diffusion in granule cell spines of the mammalian olfactory bulb.

Dendritic spines are generally believed to play a role in modulating synaptically induced electrical events. In addition, they may also confine second messengers and thus topologically limit the distance over which second messenger cascades may be functionally significant. In order to address this possibility, computer simulations of transient second messenger concentration changes were performed. The results show the importance of spine morphology and binding and extrusion mechanisms in controlling second messenger transients. In the presence of intrinsic cytoplasmic binding sites and kinetic rates similar to that expected for calcium, second messengers were confined to the spine head. In the absence of binding/extrusion mechanisms, the size and time course of the input transient to the spine head influenced the second messenger transients that might be seen at the base of the spine neck and in other spines. Large and/or sustained increases in second messenger concentration in the spine head were communicated to the spine base and to other spine heads. The results emphasize the importance of a knowledge of breakdown pathways, concentrations and kinetics of binding sites, and extrusion mechanisms for understanding the dynamics of local chemical changes for dendritic spine function.

Animals↗
PubMed1993

Terminal arborizations of olfactory nerve fibers in the glomeruli of the olfactory bulb.

The glomerulus of the olfactory bulb may serve as a fundamental organizational unit for odor representation. In this context, the axons of olfactory receptor cells with similar response spectra may converge in specific glomeruli. While the topography between the olfactory epithelium and the olfactory bulb glomeruli has been explored, the characteristics of primary afferent terminal fields within glomeruli are poorly understood. To explore this issue, reconstructions of the terminal arbors of single olfactory nerve (ON) fibers within glomeruli were carried out in the rat olfactory bulb at the light microscopic level. Tissue samples prepared with the Golgi-EM technique resulted in distinct impregnation of limited subsets of individual ON fibers. Following camera lucida reconstruction, quantitative analyses were made on selected sets of ON fibers and on the glomeruli they invaded. Most ON fibers began to arborize only after penetrating the glomeruli to a mean depth of approximately 35.9 microns. The fibers gave rise to a complex arbor of branches that was limited in total length (mean 157.7 microns) and the number of en passant varicosities or terminal enlargements (mean = 8.1). The number of varicosities and terminal boutons was proportional to the total length of branches. Also, there was a statistically significant correlation between the total branch length and the area (microns2) encompassed by the total arbor. The intraglomerular region supplied by the terminal arbor of an individual ON fiber was generally restricted. Given the recent molecular specificity attributed to olfactory receptor cells, this may provide a morphological basis for selective processing of signals encoded by specific receptors in the olfactory epithelium.

Animals↗
PubMed1993

Immunohistochemical analyses of the human olfactory bulb.

These studies explore the distribution of putative neuroactive peptides in the human olfactory bulb. Localization of synaptophysin-, serotonin-, cholecystokinin-, substance P-, and somatostatin-like staining was examined by immunocytochemical protocols. The results provide new insights into the composition and laminar segregation of subpopulations of neurons and neuronal processes in the human olfactory bulb. The prominent synaptophysin-like immunoreactivity observed in the glomeruli of the human olfactory bulb is consistent with the notion that the density of synapses, and hence the density of synaptic vesicles, is highest in the glomeruli. Serotonin-like immunoreactivity suggested a variable innervation of glomeruli ranging from a dense tangled ball of fibers within the glomerulus to a sparse innervation by a single immunoreactive fiber. There was no evidence of serotonin-like immunoreactive cell bodies in either the olfactory bulb proper, anterior olfactory nucleus, or proximal regions of the lateral olfactory tract. Cholecystokinin-like immunoreactivity was limited to fibers found largely in the juxtaglomerular region of the glomerular layer. In the deeper layers of the olfactory bulb, cholecystokinin-like immunoreactive fibers did not show any of branching or arborization that was evident in the juxtaglomerular region. Substance P-like immunoreactivity was seen in varicose fibers distributed in all of the human olfactory bulb laminae. In addition, stained multipolar neurons were found in the area of the anterior olfactory nucleus. Somatostatin-like immunoreactivity was similar to that of substance P in that a plexus of stained fibers was found in all laminae of the olfactory bulb. Also, somatostatin-like immunoreactive cell bodies were found in the area of the anterior olfactory nucleus. However, as compared to substance P, somatostatin had a less dense plexus of immunoreactive fibers in the olfactory bulb. These results increase our understanding of the fundamental organization of the human olfactory system. The current data, coupled with prior studies, provide a foundation from which to study the cellular pathology of diseases with known olfactory system sequelae such as Alzheimer's, Parkinson's, and schizophrenia.

Adult↗
PubMed1991

Localization of tyrosine hydroxylase and olfactory marker protein immunoreactivities in the human and macaque olfactory bulb.

These studies utilized specific antisera to examine the distribution and characteristics of tyrosine hydroxylase and olfactory marker protein in the olfactory bulb of the human and macaque monkey. The macaque displayed immunoreactive profiles to both antisera comparable to those described previously for other mammals. Olfactory marker protein antiserum labeled the olfactory nerve layer and glomeruli. Within the glomeruli, labeled processes were interdigitated with unlabeled processes believed to be the postsynaptic dendrites of olfactory bulb neurons. Tyrosine hydroxylase antisera labeled somata surrounding the glomeruli as well as putative dendritic processes with the glomerular neuropil. It appeared that only a subset of juxtaglomerular neurons were immunoreactive. A similar pattern was observed in the human for both antibodies. Fascicles of olfactory marker protein immunoreactive olfactory nerves often coursed long distances into the olfactory bulb prior to arborizing within a glomerulus. The data suggest that olfactory receptor cell axons destined for specific glomeruli fasciculate into bundles prior to reaching the target glomeruli. The immunoreactivity in the human to tyrosine hydroxylase was qualitatively similar to the macaque and other mammals although the number of labeled somata and intraglomerular processes appeared lower. As in the macaque, it appeared that only a subset of juxtaglomerular neurons were labeled.

Adult↗
PubMed1991

Serial reconstructions of granule cell spines in the mammalian olfactory bulb.

The morphology of olfactory bulb granule cell spines and their dendrodendritic synaptic relations with mitral and tufted cell dendrites were examined using serial electron micrographs and 3D computer reconstructions. Most granule cell spines were pedunculated with large elliptical heads and necks (stems) longer than those described for exclusively postsynaptic spines elsewhere in the nervous system. The spines typically contained a mitochondrion, which most likely reflects the metabolic requirements of the presynaptic functions of these spines. In several cases multiple spine heads were observed connected to the parent dendritic trunk via a common neck. In addition, dendritic varicosities making synaptic connections were noted. In the data set sampled, all of the reconstructions supported the hypothesis of divergence of granule cell connectivity: in no instance was a granule cell found to contact repeatedly the same mitral or tufted cell dendrite. Examination of the topological organization of reciprocal dendrodendritic synaptic connections with mitral/tufted cell dendrites revealed parallel rows of spine heads on mitral/tufted secondary dendrites separated by intervening zones of several microns in which no synaptic appositions were found. The results provide evidence regarding rules of connectivity underlying the function of local circuits in mediating lateral inhibition in the external plexiform layer of the olfactory bulb.

Animals↗